Bone morphogenetic protein pathway activation, compositions for ossification, and methods related thereto

ABSTRACT

The disclosure relates to compounds and compositions for bone formation, fracture treatment, bone grafting, bone fusion, cartilage maintenance and repair and methods related thereto. In certain embodiments, the disclosure relates to compositions comprising one or more compound(s) disclosed herein, such as clotrimazole, honokiol, magnolol, tacrolimus, pimecrolimus, sirolimus, everolimus, temsirolimus, spironolactone, fluticasone, fluticasone propionate, fluticasone furoate, linezolid, telmisartan, chlorambucil, retinol, isotretinoin, acitretin, etretinate, retinoic acid (tretinoin), teniposide, mitomycin C, cytarabine, decitabine, vinblastine, vincristine, vindesine, vinorelbine, valrubicin, doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone, plicamycin, pazopanib, topotecan, camptothecin, irinotecan, sunitinib, derivatives, or salt thereof, for use in bone growth processes. In a typical embodiment, a bone graft composition is implanted in a subject at a site of desired bone growth or enhancement.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/250,221 filed Aug. 29, 2016, which is a continuation of U.S.application Ser. No. 14/345,661 filed Mar. 19, 2014, which is theNational Stage Application of PCT/US2012/055722 filed Sep. 17, 2012,which claims priority to U.S. Provisional Application No. 61/536,168filed Sep. 19, 2011, U.S. Provisional Application No. 61/657,099 filedJun. 8, 2012, and U.S. Provisional Application No. 61/669,199 filed Jul.9, 2012. The entirety of each of these applications is herebyincorporated by reference for all purposes.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS A TEXT FILE VIA THEOFFICE ELECTRONIC FILING SYSTEM (EFS-WEB)

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 11221USCON2_ST25.txt. The text file is 9 KB, wascreated on Mar. 29, 2019, and is being submitted electronically viaEFS-Web.

FIELD

This disclosure relates to compounds and compositions for boneformation, fracture treatment, bone grafting, bone fusion, cartilagemaintenance and repair, and methods related thereto. In certainembodiments, the disclosure relates to bone graft compositionscomprising one or more compound(s) disclosed herein such asclotrimazole, honokiol, magnolol, tacrolimus, pimecrolimus, sirolimus,everolimus, temsirolimus, spironolactone, fluticasone, fluticasonepropionate, fluticasone furoate, linezolid, telmisartan, chlorambucil,retinol, isotretinoin, acitretin, etretinate, retinoic acid (tretinoin),teniposide, mitomycin C, cytarabine, decitabine, vinblastine,vincristine, vindesine, vinorelbine, valrubicin, doxorubicin,daunorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone,plicamycin, pazopanib, topotecan, camptothecin, irinotecan, sunitinib,derivatives, or salts thereof. In a typical embodiment, a bone graftcomposition is implanted in a subject at a site of desired bone growthor enhancement.

BACKGROUND

Bone grafting is typically performed for spinal fusions, after cancerousbone removal, and in certain operations, e.g., plastic surgery. Theiliac crest is often used as a donor site for autologous grafts.Complications collecting bone from the iliac crest include pain, nervedamage, hematoma and wound complications, avulsion of the anteriorsuperior iliac spine (ASIS), hematoma, herniation of the abdominalcavity contents, and cosmetic deformity. Thus, it is desirable todevelop materials and methods of forming bone that do not requireharvesting bone from remote sites of the patient.

Synthetic bone grafts typically include a matrix that holds minerals andother salts. Natural bone has an intracellular matrix mainly composed oftype I collagen, and some synthetic bone grafts include a collagenmatrix. Synthetic bone grafts typically contain bone growth factors suchas bone morphogenetic proteins (BMPs) because of their ability to induceossification in the matrix material. Recombinant human BMP-2 has beenapproved by the FDA in synthetic bone grafts such as INFUSE™. INFUSE™ isapproved for open tibial shaft fractures, lumbar interbody fusion, andsinus and alveolar ridge augmentations. However, the high cost and needfor high concentrations of BMP-2 for treatment creates a barrier forroutine clinical use. Thus, there is a need to identify additionalcompositions that can substitute or complement the use of BMPs intreating bone-related conditions.

Conflicting reports provide that sirolimus and everolimus have a bearingon osteoblast proliferation and differentiation or decreasingosteoclast-mediated bone resorption. See Kalantar-Zedah et al., CurrOpin Nephrol Hypertens., 2012, 21(4):389-403, Lee et al., Stem CellsDev., 2010, 19(4):557-68, WO2009017269, WO2005/005434 (U.S. applicationSer. No. 12/398,225), U.S. Pat. Nos. 5,258,389 and 6,015,815. Referencescited herein are not an admission of prior art.

SUMMARY

This disclosure relates to compounds and compositions for ossificationand methods related thereto. In certain embodiments, it is an object ofthe disclosure to provide certain compounds, compositions, and methodsof using these compounds to improve bone formation, fracture treatment,bone grafting, bone fusion, cartilage maintenance and repair in asubject. In a typical embodiment, the bone graft composition comprises acompound disclosed herein or derivatives that modulates BMP and/or BMPRinteractions with their natural target proteins. In specificembodiments, the disclosure relates to compounds, such as1-triarylmethyl-1H-imidazole derivatives, or salts thereof, such asclotrimazole; biphenyl-diol derivatives, or salts thereof such ashonokiol and magnolol; macrolide lactone derivatives, or salts thereofsuch as tacrolimus, pimecrolimus, sirolimus, everolimus, temsirolimus;steroid derivatives, or salts thereof such as spironolactone;3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbothioicacid derivatives, or salts thereof such as fluticasone, fluticasonepropionate, and fluticasone furoate;3-(4-morpholinophenyl)oxazolidin-2-one derivatives, or salts thereofsuch as linezolid; 1H,3′H-2,5′-bibenzo[d]imidazole derivatives or saltsthereof such as telmisartan; rentinol derivatives, or salts thereof suchas tretinoin, alitretinoin, isotretinoin, retinol, etretinate,acitretin; 4-(4-(dialkylamino)phenyl)butanoic acid derivatives, or saltsthereof such as chlorambucil; podophyllotoxin derivatives such asteniposide; aziridine derivatives such as mitomycin C; nucleosidederivatives such as cytarabine, decitabine; vinca alkaloid derivativessuch as vinorelbine, vinblastine, vincristine, and vindesine;anthracycline doxorubicin derivatives such as doxorubicin, valrubicin,daunorubicin, epirubicin, idarubicin; anthraquinone derivatives such asmitoxantrone and pixantrone; plicamycin or derivatives; pazopanib orderivatives; camptothecin or derivatives such as topotecan, irinotecan,9-aminocamptothecin; sunitinib or derivatives; and compositionsincluding such compounds, as well as their methods of use.

Examples of additional contemplated compounds include1-[(2-chlorophenyl) diphenylmethyl]-1H-imidazole, derivatives, or saltsthereof, such as clotrimazole;3′,5-di-2-propen-1-yl-[1,1′-biphenyl]-2,4′-diol, derivatives, or saltsthereof such as honokiol; 5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-3-[(1E)-2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethenyl]-14,16-dimethoxy-4,10,12,18-tetramethyl-8-(2-propen-1-yl)-15,19-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone,derivatives, or salts thereof such as tacrolimus;6,9-difluoro-11-hydroxy-16-methyl-3-oxo-17-(1-oxopropoxy)-androsta-1,4-diene-17-carbothioicacid S-(fluoromethyl) ester, derivatives, or salts thereof such asfluticasone propionate, derivatives, or salts thereof;N-[[(5S)-3-[3-fluoro-4-(4-morpholinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]-acetamide,derivatives, or salts thereof such as linezolid; and,4′-((1,7′-dimethyl-2′-propyl-1H,3′H-[2,5′-bibenzo[d]imidazol]-3′-yl)methyl)[1,1′-biphenyl]-2-carboxylicacid, derivatives, or salts thereof such as telmisartan; and9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-Hexadecahydro-9,27-dihydroxy-3-[2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone,derivatives, or salts thereof such as sirolimus, everolimus, andtemsirolimus.

Other contemplated compounds include those from the following list:capsaicin—chemical nameN-(4-hydroxy-3-methoxybenzyl)-8-methylnon-6-enamide; dihydrocapsaicin;nordihydrocapsaicin; homodihydrocapsaicin; homocapsaicin; nonivamide;diphenylcyclopropenone—chemical name 2,3-diphenylcycloprop-2-enone;estradiol—chemical name(8R,9S,13S,14S,17S)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol;ketoconazole—chemical name1-(4-(4-(((2R,4S)-2-((1H-imidazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)ethanone;ethynylestradiol—chemical name(8R,9S,13S,14S,17R)-17-ethynyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol;progesterone—chemical name(8S,9S,10R,13S,14S,17S)-17-acetyl-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3(2H)-one;salbutamol—chemical name4-(2-(tert-butylamino)-1-hydroxyethyl)-2-(hydroxymethyl)phenol;zardaverine—chemical name6-(4-(difluoromethoxy)-3-methoxyphenyl)pyridazin-3-ol; riluzole—chemicalname 6-(trifluoromethoxy)benzo[d]thiazol-2-amine;9-chloro-2-(furan-2-yl)-[1,2,4]triazolo[1,5-c]quinazolin-5-amine;prazosin—chemical name(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(furan-2-yl)methanonehydrochloride hydrate; urapidil—chemical name6-((3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dionehydrochloride; naftopidil; carmofur—chemical name5-fluoro-N-hexyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-carboxamide;5-fluoro-N-alkyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-carboxamide;fluorouracil; itraconazole—chemical name4-(4-(4-(4((2((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1-(sec-butyl)-1H-1,2,4-triazol-5(4H)-one;posaconazole; trimebutine—chemical name 2-(dimethylamino)-2-phenylbutyl3,4,5-trimethoxybenzoate; piceid—chemical name(2S,3R,4S,5S,6R)-2-(3-hydroxy-5-((E)-4-hydroxystyryl)phenoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol;resveratrol; megestrol acetate—chemical name(8R,9S,10R,13S,14S,17R)-17-acetyl-6,10,13-trimethyl-3-oxo-2,3,8,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-17-ylacetate; docetaxel—chemical name (2aR,4S,4aS,6R,9S,11R,12S,12aR,12bS)-12b-acetoxy-9-(((2R,3S)-3-((tert-butoxycarbonyl)amino)-2-hydroxy-3-phenylpropanoyl)oxy)-4,6,11-trihydroxy-4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-b]oxet-12-ylbenzoate; paclitaxel; perospirone—chemical name(3aR,7aS)-2-(4-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)butyl)hexahydro-1H-isoindole-1,3(2H)-dionehydrochloride; alprazolam—chemical name8-chloro-1-methyl-6-phenyl-4H-benzo[f][1,2,4]triazolo[4,3-a][1,4]diazepine;diazepam; oxazepam; temazepam; lorazepam; clonazepam; midazolam;fenoldopam—chemical name6-chloro-1-(4-hydroxyphenyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepine-7,8-diolmethanesulfonate; ondansetron—chemical name9-methyl-3-((2-methyl-1H-imidazol-1-yl)methyl)-2,3-dihydro-1H-carbazol-4(9H)-one;letrozole—chemical name4,4′-((1H-1,2,4-triazol-1-yl)methylene)dibenzonitrile;taxifolin—chemical name(2R,3S)-2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one;cytarabine; methyltestosterone—chemical name(8R,9S,10R,13S,14S,17S)-17-hydroxy-10,13,17-trimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3(2H)-one;artesunate—chemical name4-oxo-4-(((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-3H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)butanoicacid; artemether; dihydroartemisinin; artelinic acid; artenimol;artemotil; triclabendaxole—chemical name5-chloro-6-(2,3-dichlorophenoxy)-2-(methylthio)-1H-benzo[d]imidazole;albendazole; mebendazole; thiabendazole; fenbendazole; flubendazole;ezetimibe—chemical name(3R,4S)-1-(4-fluorophenyl)-3-((S)-3-(4-fluorophenyl)-3-hydroxypropyl)-4-(4-hydroxyphenyl)azetidin-2-one;oxiconazole—chemical name1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanoneO-(2,4-dichlorobenzyl) oxime; gebexate—chemical name ethyl4-((6-((diaminomethylene)amino) hexanoyl)oxy)benzoate;risperidone—chemical name3-(2-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)ethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one;paliperidone; bifonazole—chemical name1-([1,1′-biphenyl]-4-yl(phenyl)methyl)-1H-imidazole; indinavir;calcitriol; vinorelbine; tegaserod; irbesartan; pterostilbene;lovastatin; felodipine; daunorubicin hydrochloride; albendazole;mitoxantrone; clomid; econazole nitrate; 5-fluorouracil; includingderivatives and those substituted with one or more substituents, whichare the same or different, or salts thereof.

In certain embodiments, bone graft compositions comprise compoundsdisclosed herein and a bone morphogenetic protein and/or another growthfactor. Typically, the bone morphogenetic protein is BMP-2, BMP-5, orBMP-7. In certain embodiments, the graft composition comprises calciumphosphates and/or bone granules, hydroxyapatite and/or beta-tricalciumphosphate, alpha-tricalcium phosphate, polysaccharides or combinationsthereof. Crushed bone granules, typically obtained from the subject, areoptionally added to the graft composition.

In some embodiments, the graft contains osteogenic material can beobtained from autogenic or allogenic sources and includes, autograft,autogenic bone marrow aspirate, autogenic lipoaspirate, allogenic bonemarrow aspirate, allogenic lipoaspirate, and blends and mixturesthereof.

In some embodiments, the disclosure relates to bone graft compositionscomprising a compound disclosed herein, such as1-triarylmethyl-1H-imidazole derivatives, or salts thereof, such asclotrimazole; biphenyl-diol derivatives, or salts thereof such ashonokiol and magnolol; macrolide lactone derivatives, or salts thereofsuch as tacrolimus, pimecrolimus, sirolimus, everolimus, temsirolimus;steroid derivatives, or salts thereof such as spironolactone;3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbothioic acidderivatives, or salts thereof such as fluticasone, fluticasonepropionate, and fluticasone furoate;3-(4-morpholinophenyl)oxazolidin-2-one derivatives, or salts thereofsuch as linezolid; 1H,3′H-2,5′-bibenzo[d]imidazole derivatives or saltsthereof such as telmisartan; rentinol derivatives, or salts thereof suchas tretinoin, alitretinoin, isotretinoin, retinol, etretinate,acitretin; 4-(4-(dialkylamino)phenyl) butanoic acid derivatives, orsalts thereof such as chlorambucil, podophyllotoxin derivatives such asteniposide; aziridine derivatives such as mitomycin C; nucleosidederivatives such as cytarabine, decitabine; vinca alkaloid derivativessuch as vinorelbine, vinblastine, vincristine, and vindesine;anthracycline doxorubicin derivatives such as doxorubicin, valrubicin,daunorubicin, epirubicin, idarubicin; anthraquinone derivatives such asmitoxantrone and pixantrone; plicamycin or derivatives; pazopanib orderivatives; camptothecin or derivatives such as topotecan, irinotecan;sunitinib or derivatives or salts thereof and a graft matrix. Typically,the matrix comprises a collagen sponge and/or a compression resistanttype I collagen and calcium phosphates. In other embodiments, the matrixis a hydrogel.

In certain embodiments, the disclosure contemplates a graft compositionor matrix comprising compounds disclosed herein such as tacrolimus,pimecrolimus, sirolimus, everolimus, temsirolimus, teniposide, mitomycinC, cytarabine, decitabine, vinblastine, vincristine, vindesine,vinorelbine, valrubicin, doxorubicin, daunorubicin, epirubicin,idarubicin, mitoxantrone, pixantrone, plicamycin, pazopanib, topotecan,camptothecin, irinotecan, sunitinib, wherein the graft matrix is acollagen or demineralized bone matrix or ceramic of other scaffolddisclosed herein without exogenous cells.

In some embodiments, the disclosure relates to kits comprising a graftcomposition, a compound disclosed herein, and a graft matrix. In certainembodiments, the kits further comprise a bone morphogenetic proteinand/or another growth factor. In certain embodiments, the kits furthercomprise a transfer device, such as a syringe or pipette.

In some embodiments, the disclosure relates to methods of generatingBMP-mediated osteoblasts comprising administering an effective amount ofcompound(s) disclosed herein to cells capable of osteoblasticdifferentiation, such as mesenchymal stem cells and pre-osteoblasticcells.

In some embodiments, the disclosure relates to methods of forming boneor cartilage, comprising implanting a graft composition comprising acompound disclosed herein, such as 1-triarylmethyl-1H-imidazolederivatives, or salts thereof, such as clotrimazole; biphenyl-diolderivatives, or salts thereof such as honokiol and magnolol; macrolidelactone derivatives, or salts thereof such as tacrolimus, pimecrolimus,sirolimus, everolimus, temsirolimus; steroid derivatives, or saltsthereof such as spironolactone;3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbothioicacid derivatives, or salts thereof such as fluticasone, fluticasonepropionate, and fluticasone furoate;3-(4-morpholinophenyl)oxazolidin-2-one derivatives, or salts thereofsuch as linezolid; 1H,3′H-2,5′-bibenzo[d]imidazole derivatives or saltsthereof such as telmisartan; rentinol derivatives, or salts thereof suchas tretinoin, alitretinoin, isotretinoin, retinol, etretinate,acitretin; 4-(4-(dialkylamino)phenyl)butanoic acid derivatives, or saltsthereof such as chlorambucil, podophyllotoxin derivatives such asteniposide; aziridine derivatives such as mitomycin C; nucleosidederivatives such as cytarabine, decitabine; vinca alkaloid derivativessuch as vinorelbine, vinblastine, vincristine, and vindesine;anthracycline doxorubicin derivatives such as doxorubicin, valrubicin,daunorubicin, epirubicin, idarubicin; anthraquinone derivatives such asmitoxantrone and pixantrone; plicamycin or derivatives; pazopanib orderivatives; camptothecin or derivatives such as topotecan, irinotecan;sunitinib or derivatives or salts thereof in a subject under conditionssuch that bone or cartilage forms in the graft. Typically, the subjecthas a void in the bony structure wherein the graft composition isimplanted in the void. In certain embodiments, the void is in a boneselected from an extremity, maxilla, mandible, pelvis, spine and/orcranium. In certain embodiments, the void is a result of surgicalremoval of bone. In certain embodiments, the void is between bone and animplanted medical device. In another embodiment, the method furthercomprises the step of securing movement of bone structure with afixation system, and removing the system after bone forms in theimplanted graft.

In certain embodiments, the disclosure contemplates local bone formationfor fracture repair, segmental bone defects, spine fusion, bonegrafting, and regional bone enhancement for osteopenic bones before theyfracture (e.g. hip, vertebral body, etc) by deliver locally to inducelocal bone formation.

In certain embodiments, the disclosure relates to methods of growingbone in subject by locally administering, such as by injection, acomposition comprising a compound disclosed herein, optionally incombination with a growth factor, about the area of desired bone growth.In certain embodiments, the disclosure relates to methods of growingbone comprising administering a pharmaceutical composition comprisingclotrimazole, honokiol, magnolol, tacrolimus, pimecrolimus, sirolimus,everolimus, temsirolimus, spironolactone, fluticasone, fluticasonepropionate, fluticasone furoate, linezolid, telmisartan, chlorambucil,retinol, isotretinoin, acitretin, etretinate, retinoic acid (tretinoin),teniposide, mitomycin C, cytarabine, decitabine, vinblastine,vincristine, vindesine, vinorelbine, valrubicin, doxorubicin,daunorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone,plicamycin, pazopanib, topotecan, camptothecin, irinotecan, sunitinib,derivatives, or pharmaceutically acceptable salts thereof to a subjectin an area of desired growth, wherein the administration is localizeddirectly about the area of desired growth. In certain embodiments, theadministration is not oral administration. In certain embodiments, theadministration is through a catheter or hypodermic needle with a tipthat is not in a vein. In certain embodiments, the administration is byinjection into the subcutaneous tissue or in or about an area typicallyoccupied by bone or between vertebra, e.g., in the area usually occupiedby in the intervertebral disc, to form a spinal fusion.

In certain embodiments, the method contemplates implanting a graftcomposition in a desired area of the subject and locally administering acomposition comprising a compound disclosed herein, optionally incombination with a growth factor, in the graft or about the area of thegraft implant such as by injection.

In certain embodiments, the disclosure relates to uses of compoundsdisclosed herein for cartilage regeneration e.g., between intervertebraldisc and articular, jaw, elbow, knee, ankle, wrist, and hip joints.Methods contemplate oral administration, intravenous administration, ordirect injection at the desired site(s) of the subject.

In some embodiments, the disclosure relates to methods of performingspinal fusion comprising implanting a bone graft composition comprisinga compound disclosed herein, such as 1-triarylmethyl-1H-imidazolederivatives, or salts thereof, such as clotrimazole; biphenyl-diolderivatives, or salts thereof such as honokiol and magnolol; macrolidelactone derivatives, or salts thereof such as tacrolimus, pimecrolimus,sirolimus, everolimus, temsirolimus; steroid derivatives, or saltsthereof such as spironolactone;3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbothioic acid derivatives, or salts thereof suchas fluticasone, fluticasone propionate, and fluticasone furoate;3-(4-morpholinophenyl)oxazolidin-2-one derivatives, or salts thereofsuch as linezolid; 1H,3′H-2,5′-bibenzo[d]imidazole derivatives or saltsthereof such as telmisartan; rentinol derivatives, or salts thereof suchas tretinoin, alitretinoin, isotretinoin, retinol, etretinate,acitretin; 4-(4-(dialkylamino)phenyl)butanoic acid derivatives, or saltsthereof such as chlorambucil, podophyllotoxin derivatives such asteniposide; aziridine derivatives such as mitomycin C; nucleosidederivatives such as cytarabine, decitabine; vinca alkaloid derivativessuch as vinorelbine, vinblastine, vincristine, and vindesine;anthracycline doxorubicin derivatives such as doxorubicin, valrubicin,daunorubicin, epirubicin, idarubicin; anthraquinone derivatives such asmitoxantrone and pixantrone; plicamycin or derivatives; pazopanib orderivatives; camptothecin or derivatives such as topotecan, irinotecan;sunitinib or derivatives or salts thereof configured to grow bonebetween two vertebrae of a subject. In certain embodiments, thecomposition further comprises a bone morphogenetic protein and/oranother growth factor. In a typical embodiment, the subject is diagnosedwith degenerative disc disease or has symptoms of back pain.

In some embodiments, the disclosure relates to methods of inserting aprosthetic device or anchor, comprising exposing a bone and implanting agraft composition comprising a compound disclosed herein in contact withthe bone. In certain embodiments, one implants the prosthetic device oranchor in the graft composition. In certain embodiments, the compositionfurther comprises a bone morphogenetic protein and/or another growthfactor.

In some embodiments, the disclosure relates to pharmaceuticalcompositions comprising compounds disclosed herein or pharmaceuticallyacceptable salts thereof. In certain embodiments, the compositionfurther comprises a bone morphogenetic protein and/or another growthfactor. In certain embodiments, the pharmaceutical composition isformulated to release over a 12 hour, 1 day, 3 day, 5 day, 7 day, twoweek, or one month period.

In certain embodiments, the disclosure relates to methods of preventingor treating a bone fracture, comprising administering a pharmaceuticalcomposition comprising a compound disclosed herein, such as1-triarylmethyl-1H-imidazole derivatives, or salts thereof, such asclotrimazole; biphenyl-diol derivatives, or salts thereof such ashonokiol and magnolol; macrolide lactone derivatives, or salts thereofsuch as tacrolimus, pimecrolimus, sirolimus, everolimus, temsirolimus;steroid derivatives, or salts thereof such as spironolactone;3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbothioic acidderivatives, or salts thereof such as fluticasone, fluticasonepropionate, and fluticasone furoate; 3-(4-morpholinophenyl)oxazolidin-2-one derivatives, or salts thereof such as linezolid;1H,3′H-2,5′-bibenzo[d]imidazole derivatives or salts thereof such astelmisartan; rentinol derivatives, or salts thereof such as tretinoin,alitretinoin, isotretinoin, retinol, etretinate, acitretin;4-(4-(diethylamino) phenyl)butanoic acid derivatives, or salts thereofsuch as chlorambucil; podophyllotoxin derivatives such as teniposide;aziridine derivatives such as mitomycin C; nucleoside derivatives suchas cytarabine, decitabine; vinca alkaloid derivatives such asvinorelbine, vinblastine, vincristine, and vindesine; anthracyclinedoxorubicin derivatives such as doxorubicin, valrubicin, daunorubicin,epirubicin, idarubicin; anthraquinone derivatives such as mitoxantroneand pixantrone; plicamycin or derivatives; pazopanib or derivatives;camptothecin or derivatives such as topotecan, irinotecan; sunitinib orderivatives or salts thereof or a pharmaceutically acceptable saltthereof, to a subject at risk for, exhibiting symptoms of, or diagnosedwith a bone fracture. In certain embodiments, the composition furthercomprises a bone morphogenetic protein and/or another growth factor.

In certain embodiments, the administration is localized. In certainembodiments, administration is achieved through oral delivery,intravenous delivery, parenteral delivery, intradermal delivery,percutaneous delivery, or subcutaneous delivery. In some embodiments,the method further comprises the step of exposing the bone fracture topulsed electromagnetic fields. In further embodiments, the subject isdiagnosed with a long bone shaft fracture such as a tibia or femurfracture corrected with intramedullary nail fixation.

In some embodiments, the disclosure relates to methods of preventing ortreating a bone degenerative disease, comprising administering apharmaceutical composition comprising a compound disclosed herein, suchas 1-triarylmethyl-1H-imidazole derivatives, or salts thereof, such asclotrimazole; biphenyl-diol derivatives, or salts thereof such ashonokiol and magnolol; macrolide lactone derivatives, or salts thereofsuch as tacrolimus, pimecrolimus, sirolimus, everolimus, temsirolimus;steroid derivatives, or salts thereof such as spironolactone;3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbothioic acidderivatives, or salts thereof such as fluticasone, fluticasonepropionate, and fluticasone furoate; 3-(4-morpholinophenyl)oxazolidin-2-one derivatives, or salts thereof such as linezolid;1H,3′H-2,5′-bibenzo[d]imidazole derivatives or salts thereof such astelmisartan; rentinol derivatives, or salts thereof such as tretinoin,alitretinoin, isotretinoin, retinol, etretinate, acitretin;4-(4-(diethylamino)phenyl) butanoic acid derivatives, or salts thereofsuch as chlorambucil; podophyllotoxin derivatives such as teniposide;aziridine derivatives such as mitomycin C; nucleoside derivatives suchas cytarabine, decitabine; vinca alkaloid derivatives such asvinorelbine, vinblastine, vincristine, and vindesine; anthracyclinedoxorubicin derivatives such as doxorubicin, valrubicin, daunorubicin,epirubicin, idarubicin; anthraquinone derivatives such as mitoxantroneand pixantrone; plicamycin or derivatives; pazopanib or derivatives;camptothecin or derivatives such as topotecan, irinotecan; sunitinib orderivatives or salts thereof or a pharmaceutically acceptable saltsthereof, to a subject at risk for, exhibiting symptoms of, or diagnosedwith a bone degenerative disease. In certain embodiments, thecomposition further comprises a bone morphogenetic protein and/oranother growth factor. In certain embodiments, the administration issystemic, or administration is achieved through oral delivery,intravenous delivery, parenteral delivery, intradermal delivery,percutaneous delivery, or subcutaneous delivery. In some embodiments,the disease is osteoporosis, osteitis deformans, bone metastasis,multiple myeloma, primary hyperparathyroidism, or osteogenesisimperfecta.

In some embodiments, the disclosure relates to methods for decreasingthe time required to form new bone in the presence of a bonemorphogenetic protein, comprising co-administering at least one compounddisclosed herein, such as 1-triarylmethyl-1H-imidazole derivatives, orsalts thereof, such as clotrimazole; biphenyl-diol derivatives, or saltsthereof such as honokiol and magnolol; macrolide lactone derivatives, orsalts thereof such as tacrolimus, pimecrolimus, sirolimus, everolimus,temsirolimus; steroid derivatives, or salts thereof such asspironolactone; 3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbothioic acidderivatives, or salts thereof such as fluticasone, fluticasonepropionate, and fluticasone furoate;3-(4-morpholinophenyl)oxazolidin-2-one derivatives, or salts thereofsuch as linezolid; 1H,3′H-2,5′-bibenzo[d]imidazole derivatives or saltsthereof such as telmisartan; rentinol derivatives, or salts thereof suchas tretinoin, alitretinoin, isotretinoin, retinol, etretinate,acitretin; 4-(4-(dialkylamino)phenyl)butanoic acid derivatives, or saltsthereof such as chlorambucil; podophyllotoxin derivatives such asteniposide; aziridine derivatives such as mitomycin C; nucleosidederivatives such as cytarabine, decitabine; vinca alkaloid derivativessuch as vinorelbine, vinblastine, vincristine, and vindesine;anthracycline doxorubicin derivatives such as doxorubicin, valrubicin,daunorubicin, epirubicin, idarubicin; anthraquinone derivatives such asmitoxantrone and pixantrone; plicamycin or derivatives; pazopanib orderivatives; camptothecin or derivatives such as topotecan, irinotecan;sunitinib or derivatives or salts thereof and another active ingredient.

In some embodiments, the disclosure relates to a process for engineeringbone tissue comprising combining a compound disclosed herein, such as1-triarylmethyl-1H-imidazole derivatives, or salts thereof, such asclotrimazole; biphenyl-diol derivatives, or salts thereof such ashonokiol and magnolol; macrolide lactone derivatives, or salts thereofsuch as tacrolimus, pimecrolimus, sirolimus, everolimus, temsirolimus;steroid derivatives, or salts thereof such as spironolactone;3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbothioic acidderivatives, or salts thereof such as fluticasone, fluticasonepropionate, and fluticasone furoate;3-(4-morpholinophenyl)oxazolidin-2-one derivatives, or salts thereofsuch as linezolid; 1H,3′H-2,5′-bibenzo[d]imidazole derivatives or saltsthereof such as telmisartan; rentinol derivatives, or salts thereof suchas tretinoin, alitretinoin, isotretinoin, retinol, etretinate,acitretin; 4-(4-(dialkylamino)phenyl)butanoic acid derivatives, or saltsthereof such as chlorambucil; podophyllotoxin derivatives such asteniposide; aziridine derivatives such as mitomycin C; nucleosidederivatives such as cytarabine, decitabine; vinca alkaloid derivativessuch as vinorelbine, vinblastine, vincristine, and vindesine;anthracycline doxorubicin derivatives such as doxorubicin, valrubicin,daunorubicin, epirubicin, idarubicin;

anthraquinone derivatives such as mitoxantrone and pixantrone;plicamycin or derivatives; pazopanib or derivatives; camptothecin orderivatives such as topotecan, irinotecan; sunitinib or derivatives orsalts thereof and optionally a bone morphogenetic protein, with a cellselected from the group consisting of osteogenic cells, pluripotent stemcells, mesenchymal cells, and embryonic stem cells.

In certain embodiments, the disclosure relates to using1-triarylmethyl-1H-imidazole derivatives, or salts thereof, such asclotrimazole; biphenyl-diol derivatives, or salts thereof such ashonokiol and magnolol; macrolide lactone derivatives, or salts thereofsuch as tacrolimus, pimecrolimus, sirolimus, everolimus, temsirolimus;steroid derivatives, or salts thereof such as spironolactone;3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbothioic acidderivatives, or salts thereof such as fluticasone, fluticasonepropionate, and fluticasone furoate;3-(4-morpholinophenyl)oxazolidin-2-one derivatives, or salts thereofsuch as linezolid; 1H,3′H-2,5′-bibenzo[d]imidazole derivatives or saltsthereof such as telmisartan; rentinol derivatives, or salts thereof suchas tretinoin, alitretinoin, isotretinoin, retinol, etretinate,acitretin; 4-(4-(dialkylamino)phenyl)butanoic acid derivatives, or saltsthereof such as chlorambucil; podophyllotoxin derivatives such asteniposide; aziridine derivatives such as mitomycin C; nucleosidederivatives such as cytarabine, decitabine; vinca alkaloid derivativessuch as vinorelbine, vinblastine, vincristine, and vindesine;anthracycline doxorubicin derivatives such as doxorubicin, valrubicin,daunorubicin, epirubicin, idarubicin; anthraquinone derivatives such asmitoxantrone and pixantrone; plicamycin or derivatives; pazopanib orderivatives; camptothecin or derivatives such as topotecan, irinotecan;sunitinib or derivatives or salts thereof in the production of amedicament for the treatment or prevention of a bone disease or otherapplications disclosed herein.

In certain embodiments, the disclosure relates to a bone graftcomposition comprising a bone growth-inducing amount of1-triarylmethyl-1H-imidazole derivatives, or salts thereof, such asclotrimazole; biphenyl-diol derivatives, or salts thereof such ashonokiol and magnolol; macrolide lactone derivatives, or salts thereofsuch as tacrolimus, pimecrolimus, sirolimus, everolimus, temsirolimus;steroid derivatives, or salts thereof such as spironolactone;3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbothioicacid derivatives, or salts thereof such as fluticasone, fluticasonepropionate, and fluticasone furoate;3-(4-morpholinophenyl)oxazolidin-2-one derivatives, or salts thereofsuch as linezolid; 1H,3′H-2,5′-bibenzo[d]imidazole derivatives or saltsthereof such as telmisartan; rentinol derivatives, or salts thereof suchas tretinoin, alitretinoin, isotretinoin, retinol, etretinate,acitretin; 4-(4-(diethylamino)phenyl) butanoic acid derivatives, orsalts thereof such as chlorambucil; podophyllotoxin derivatives such asteniposide; aziridine derivatives such as mitomycin C; nucleosidederivatives such as cytarabine, decitabine; vinca alkaloid derivativessuch as vinorelbine, vinblastine, vincristine, and vindesine;anthracycline doxorubicin derivatives such as doxorubicin, valrubicin,daunorubicin, epirubicin, idarubicin; anthraquinone derivatives such asmitoxantrone and pixantrone; plicamycin or derivatives; pazopanib orderivatives; camptothecin or derivatives such as topotecan, irinotecan;sunitinib or derivatives or salts thereof and a pharmaceuticallyacceptable carrier.

In certain embodiments, the derivative is 1-triarylmethyl-1H-imidazoleor salt thereof with one or more substituents.

In certain embodiments, the derivative is 1,1′-biphenyl-2,4′-diol orsalt thereof with one or more substituents.

In certain embodiments, the derivative is5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-3-[(1E)-2-[cyclohexyl]ethenyl]-15,19-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetroneor salt thereof with one or more substituents.

In certain embodiments, the derivative is3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbothioicacid or salt thereof with one or more substituents.

In certain embodiments, the derivative is1,6,7,8,9,10,11,12,13,14,15,16-dodecahydro-3′H-spiro[cyclopenta[a]phenanthrene-17,2′-furan]-3,5′(2H,4′H)-dioneor salt thereof with one or more substituents.

In certain embodiments, the derivative is3-(4-morpholinophenyl)oxazolidin-2-one or salt thereof with one or moresubstituents.

In certain embodiments, the derivative is1-(octa-1,3,5,7-tetraen-1-yl)cyclohex-1-ene orocta-1,3,5,7-tetraen-1-ylbenzene or salt thereof with one or moresubstituents.

In certain embodiments, the derivative is3′-([1,1′-biphenyl]-4-ylmethyl)-1H,3′H-2,5′-bibenzo[d]imidazole or saltthereof with one or more substituents.

In certain embodiments, the derivative is4-(4-(dialkylamino)phenyl)butanoic acid or salt thereof with one or moresubstituents.

In certain embodiments, the 1-triarylmethyl-1H-imidazole derivative isclotrimazole or salt thereof optionally substituted with one or moresubstituents.

In certain embodiments, the biphenyl-diol derivative is honokiol ormagnolol or salt thereof optionally substituted with one or moresubstituents.

In certain embodiments, the macrolide lactone derivative is tacrolimus,pimecrolimus, sirolimus, everolimus, temsirolimus or salt thereofoptionally substituted with one or more substituents.

In certain embodiments, the3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbothioicacid derivative is fluticasone, fluticasone propionate, or fluticasonefuroate or salt thereof optionally substituted with one or moresubstituents.

In certain embodiments, the 3-(4-morpholinophenyl)oxazolidin-2-onederivative is linezolid or salt thereof optionally substituted with oneor more substituents.

In certain embodiments, the 1H,3′H-2,5′-bibenzo[d]imidazole derivativeis telmisartan or salt thereof optionally substituted with one or moresubstituents.

In certain embodiments, the steroid derivative is spironolactone or saltthereof optionally substituted with one or more substituents.

In certain embodiments, the retinol derivative is tretinoin,alitretinoin, isotretinoin, retinol, etretinate, acitretin or saltthereof optionally substituted with one or more substituents.

In certain embodiments, the 4-(4-(dialkylamino)phenyl)butanoic acidderivative is chlorambucil or salt thereof with one or moresubstituents.

In certain embodiments, the podophyllotoxin is5-phenyl-9-((2-(thiophen-2-yl)hexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-5,5a,8a,9-tetrahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-6(8H)-oneor derivative such as teniposide optionally substituted with one or moresubstituents.

In certain embodiments, the aziridine is1,1a,2,8,8a,8b-hexahydroazirino[2′,3′:3,4]pyrrolo[1,2-a]indole-4,7-dioneor derivative such as mitomycin C optionally substituted with one ormore substituents.

In certain embodiments, the nucleoside is1-(5-methyltetrahydrofuran-2-yl)pyrimidin-2(1H)-one or derivative suchas cytarabine, decitabine optionally substituted with one or moresubstituents.

In certain embodiments, the vinca alkaloid is9-(2,4,5,6,7,8,9,10-octahydro-1H-3,7-methano[1]azacycloundecino[5,4-b]indol-9-yl)-3a,3a1,4,5,5a,6,11,12-octahydro-1H-indolizino[8,1-cd]carbazoleor derivative such as vinorelbine, vinblastine, vincristine, andvindesine optionally substituted with one or more substituents.

In certain embodiments, the anthracycline is8-acetyl-10-((4-amino-5-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-6,8,11-trihydroxy-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dioneor derivative such as doxorubicin, valrubicin, daunorubicin, epirubicin,idarubicin optionally substituted with one or more substituents.

In certain embodiments, the anthraquinone is anthracene-9,10-dione orderivative such as mitoxantrone and pixantrone optionally substitutedwith one or more substituents.

In certain embodiments, plicamycin or derivatives are optionallysubstituted with one or more substituents.

In certain embodiment, pazopanib or derivatives are optionallysubstituted with one or more substituents.

In certain embodiments, the camptothecin is1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione orderivatives such as topotecan and irinotecan optionally substituted withone or more substituents.

In certain embodiments, sunitinib or derivatives are optionallysubstituted with one or more substituents.

In certain embodiments, the bone morphogenetic protein is selected fromthe group consisting BMP-2, BMP-5, BMP-6, BMP-7, BMP-9, and combinationsthereof.

In certain embodiments, the compounds described herein may be usedlocally such as injection percutaneously at any bone formation site(fracture, spine fusion delayed a day or several days after surgery). Incertain embodiments, the compounds may also be bound to a matrix orscaffold and delivered with growth factors, cells (MSCs or others), oron a dry carrier matrix to direct local bone formation in the shape ofthe carrier/scaffold. Within certain embodiments, it is alsocontemplated that one or more of these compounds disclosed herein may beused alone or in combination with multiple compounds, with or withoutexogenous growth factors, and/or in combination with other promotingagents of the BMP pathway such as a noggin inhibitor, a Smurf inhibitorand/or a JAB1 inhibitor.

In certain embodiments, the disclosure contemplates delivery ofcompositions comprising compounds disclosed herein via a liquid orflowable gel optionally including collagen, hydroxyapatite,demineralized bone, or polymer matrix or others, e.g., as disclosedherein. In certain embodiments, the compositions are injected into thecentral cavity or interstices of a structural element such as a bonecage made from allograft, polymer thermoplastic, metal such as titaniumor aluminum, polyether ether ketone (PEEK) such as those generated from4,4′-difluorobenzophenone, or other polymer.

In certain embodiments, the disclosure contemplates bone graftscontaining more than 10, 15, 20, 25, 30, 35 mM of a compound disclosedherein such as a macrolide lactone derivative such as tacrolimus,pimecrolimus, sirolimus, everolimus, temsirolimus per about 100 mm³, orabout 150 mm³, or 50 to 200 mm³, or about 100 to 200 mm³ of bone graftvolume. In some embodiments, the disclosure contemplates more than 10 mMor 20 mM of sirolimus. In some embodiments, the disclosure contemplatesmore than 10 mM or 15 mM of everolimus. In some embodiments, thedisclosure contemplates more than 10 mM or 15 mM of tacrolimus. Incertain embodiments, the bone graft is contemplated for all usesdisclose herein. In certain embodiments, the subject is human.

In certain embodiments, the disclosure contemplates bone graftcontaining more than 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.8, 2.0, or 3.0 mgof a macrolide lactone derivative such as tacrolimus, pimecrolimus,sirolimus, everolimus, temsirolimus, per about 100 mm³, or about 150mm³, or 50 to 200 mm³, or about 100 to 200 mm³ of bone graft volume. Insome embodiments, the disclosure contemplates more than 1.5 mg ofsirolimus. In some embodiments, the disclosure contemplates more that0.9 mg of everolimus. In some embodiments, the disclosure contemplatesmore that 0.7 mg of tacrolimus. In certain embodiments, the bone graftis contemplated for all uses disclose herein. In certain embodiments,the subject is human.

In certain embodiments, the disclosure contemplates kit containing abone graft and a composition comprising more than 0.7, 0.8, 0.9, 1.0,1.2, 1.5, 1.8, 2.0, or 3.0 mg of a macrolide lactone derivative such astacrolimus, pimecrolimus, sirolimus, everolimus, temsirolimus per about100, 125, or 150 mm³ of bone graft volume. In some embodiments, thedisclosure contemplates more than 1.5 mg of sirolimus. In someembodiments, the disclosure contemplates more that 0.9 mg of everolimus.In some embodiments, the disclosure contemplates more that 0.7 mg oftacrolimus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the BMP signaling pathway. Although it is notintended that embodiments of the disclosure be limited by any particularmechanism, it is believed that the embodiments may act through the BMPpathway. In the BMP pathway, BMP binds to the BMP receptor type II thatin turn activates BMPR-1. Activated BMPR-1 phosphorylates receptorregulated Smads (Smad1/5/8) which form complexes with Smad4 facilitatingnuclear entry of Smad complexes. Activated Smad complexes regulate geneexpression of BMP-responsive genes. Extracellularly, Noggin antagonizessignaling by binding to BMP and thus inhibiting binding of BMP to itsreceptor complex.

FIG. 2 illustrates the structural features of BMP-Receptor interactionsand important amino acids that determine binding specificity andaffinity. Illustrative structures of BMP-2, BMPR-IA, and BMPR-II aredepicted. Important aminoacids are labeled in ‘yellow’ color.

FIG. 3 shows data for the activity of test compounds in the ALP assay.Capsaicin is SNGP-003-01 H04; diphenylcyclopropenone is SNGP-003-01 E05;clotrimazole is SNGP-003-01 C06; beta-estradiol is SNGP-003-01 B09;ketoconazole or1-(4-(4-(((2R,4S)-2((1H-imidazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)ethanoneis SNGP-003-01 A10; and ethynylestradiol is SNGP-003-01 E10.

FIG. 4 shows data on the activity of test compounds in a BMP-Noggincompetitive ALP assay. Progesterone is SNGP-003-01 H03; capsaicin isSNGP-003-01 H04; salbutamol sulfate is SNGP-003-01 A05; Zardaverine isSNGP-003-01 H05; clotrimazole is SNGP-003-01 C06; Riluzole isSNGP-003-01 F06; (CGS 15943)9-chloro-2-(furan-2-yl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine isSNGP-003-01 B07; Prazosin is SNGP-003-01 G07; and urapidil hydrochlorideis SNGP-003-01 H07. (CGS 15943)9-chloro-2-(furan-2-yl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine andCapsaicin caused cell lifting from culture plates as determined based onlow cell number and low yield of protein after cell harvesting and celllysis.

FIG. 5 shows data suggesting dose dependent increase of BMP-induced ALPactivity by nanomolar concentrations of clotrimazole.

FIG. 6 shows data for the activity of test compounds in the ALP assay.Docetaxel is SNGP-003-02 A06; honokiol is SNGP-003-02 B06; carmofur isSNGP-003-02 D06; Methyltestosterone is SNGP-003-02 A07; itraconazole isSNGP-003-02 F07; triclabendazole is SNGP-003-02 C08; flubendazole isSNGP-003-02 A09; tacrolimus is SNGP-003-02 B09; and trimebutine maleateis SNGP-003-02 F09. Docetaxel, carmofur, itraconazole, and flubendazolecaused cell lifting from culture plates.

FIG. 7 shows data of activity for test compounds in a BMP-Noggincompetitive ALP assay. Ketoconazole is SNGP-003-01 A10; ethynylestradiolis SNGP-003-01 E10; cytarabine is SNGP-003-01 F10; taxifolin isSNGP-003-02 E03; letrozole is SNGP-003-02 C04; ondansetron isSNGP-003-02 F04; alprazolam is SNGP-003-02 A05; perospirone isSNGP-003-02 H05; docetaxel is SNGP-003-02 A06; and honokiol isSNGP-003-02 B06. Ketoconazole and cytarabine caused cell lifting fromculture plates.

FIG. 8 shows data on the activity of test compounds in a BMP-Noggincompetitive ALP Assay. Methyltestosterone is SNGP-003-02 A07; artesunateis SNGP-003-02 E07; triclabendazole is SNGP-003-02 C08; ezetimibe isSNGP-003-02 F08; tacrolimus is SNGP-003-02 B09; trimebutine maleate isSNGP-003-02 F09; megestrol acetate is SNGP-003-02 E10; and oxiconazolenitrate is SNGP-003-02 B11. Artesunate and oxiconazole nitrate causedcell lifting from culture plates.

FIG. 9 shows data on the activity of test compounds in an ALP assay.Oxiconazole nitrate is SNGP-003-02 B11; pioglitazone hydrochloride isSNGP-003-03 E04; zolpidem tartarate is SNGP-003-03 D05; fenoldopammesylate is SNGP-003-03 H05; 2′,3′-dideoxyinosine is SNGP-003-03 E06;calcipotriol is SNGP-003-03 G06; icariin is SNGP-003-03 E07;rosiglitazole hydrochloride is SNGP-003-03 H07; oligomycin isSNGP-003-03 B08; rolipram is SNGP-003-03 G08; fluticasone propionate isSNGP-003-03 G10; indinavir sulphate is SNGP-003-03H10; midazolamhydrochloride is SNGP-003-03 A11; bifonazole is SNGP-003-04 C02;calcitriol is SNGP-003-04 C03; vinorelbinee bitatrate is SNGP-003-04B04; linezolid is SNGP-003-04 C04; irbesartan is SNGP-003-04 F04; andpterostilbene is SNGP-003-04 A05. Calcipotriol, calcitriol, andvinorelbinee bitatrate caused cell lifting from culture plates.

FIG. 10 shows data on the activities in ALP assays for select compoundsat 250 nM concentration. SVAK12X is cycloguanil-chemical name1-(4-chlorophenyl)-2,2-dimethyl-1,3,5-triazine-4,6-diamine.

FIG. 11 shows data on the activity of test compounds in a BMP-Noggincompetitive ALP assay. Telmisartan is SNGP-003-04H08;

FIG. 12 shows data on the activity of test compounds in an ALP assay.Retinoic acid is SNGP-201-01-006; spironolactone is SNGP-201-01D06;5-fluorouracil is SNGP-201-01-H02; 3,5,3′-triiodothryonine isSNGP-201-01-A08; econazole nitrate is SNGP-201-01-E09; oxytetracyclinehydrochloride is SNGP-201-01-H11. Econazole nitrate and 5-fluorouracilcaused cell lifting from culture plates.

FIG. 13 shows data on the activity of test compounds in an ALP assay.Chlorambucil is SNGP-201-02-E05; isotretinoin is SNGP-201-03-E05;albendazole is SNGP-201-03-A03; mitoxantrone is SNGP-201-03-B04; clomidis SNGP-201-03-A05. Albendazole, mitoxantrone, and clomid caused celllifting from culture plates.

FIG. 14 shows data of activity for test compounds in a BMP-Noggincompetitive ALP assay. Isotretinoin is SNGP-201-03-E05.

FIG. 15 shows data on the activity of test compounds in an ALP assay.Isotretinoin is SNGP-201-03-E05 and acitretin is SNGP-201-04-E06.Daunorubicin hydrochloride is SNGP-201-04-E03. Daunorubicinhydrochloride caused cell lifting from culture plates.

FIG. 16 shows data of activity for test compounds in a BMP-Noggincompetitive ALP assay. Acitretin is SNGP-201-04-E06; lovastatin isSNGP-201-04-005; and felodipine is SNGP-201-04-006. Lovastatin andfelodipine caused cell lifting from culture plates.

FIG. 17 shows data of activity for macrolide analogs in an ALP activityassay. Vertical axis (Y-axis) is relative ALP activity, and thehorizontal axis (X-axis) is treatment.

DETAILED DISCUSSION Terms

“Ossification” refers to the process of laying down new bone by cellscalled osteoblasts. The term includes the growth in healing bonefractures treated by cast or by open reduction and stabilization bymetal plate and screws. Ossification can also result in the formation ofbone tissue in an extraskeletal location.

The term “bone morphogenetic protein” or “BMP” refers to any one of thefamily of growth factors or fragments thereof with the ability to inducethe formation of bone and/or cartilage. The BMP receptors are typicallyserine-threonine kinases. It is not intended that BMP refer to anyparticular protein sequence and may or may not have certainglycosylation patterns attached thereto provided that the molecule hassufficient structural homology to any one of the known BMPs describedbelow and retains some functional ability to promote bone growth,cartilage growth, or osteoblast differentiation. BMPs may be isolatedfrom natural or non-natural sources, such as, but not limited to,recombinant or synthetic methods. References to BMPs generally or aspecific BMP, e.g, BMP-2, includes recombinant or synthetically isolatedversions unless otherwise provide for herein. Combinations of BMPs arecontemplated. BMP-2 is known to induce bone and cartilage formation andplay a role in osteoblast differentiation. BMP-3 is known to induce boneformation. BMP-4 is known to regulate the formation of teeth, limbs andbone from mesoderm and play a role in fracture repair. BMP-5 is known tofunction in cartilage development. BMP-6 is known to play a role injoint integrity and bone formation/repair. BMP-7 and BMP-9 are known toplay a role in osteoblast differentiation. BMP-1 is a knownmetalloprotease that acts on procollagen I, II, and III and is involvedin cartilage development.

As used herein, the term “derivative” refers to a structurally similarcompound that retains sufficient functional attributes of the identifiedanalogue. The derivative may be structurally similar because it islacking one or more atoms, substituted, a salt, in differenthydration/oxidation states, or because one or more atoms within themolecule are switched, such as, but not limited to, replacing an oxygenatom with a sulfur atom or replacing an amino group with a hydroxygroup. The derivative may be a prodrug. Derivatives can be prepare byany variety of synthetic methods or appropriate adaptations presented insynthetic or organic chemistry text books, such as those provide inMarch's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, Wiley, 6th Edition (2007) Michael B. Smith or DominoReactions in Organic Synthesis, Wiley (2006) Lutz F. Tietze.

The term “substituted” refers to a molecule wherein at least onehydrogen atom is replaced with a substituent. When substituted, one ormore of the groups are “substituents.” The molecule may be multiplysubstituted. In the case of an oxo substituent (“═O”), two hydrogenatoms are replaced. Example substituents within this context may includehalogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl,carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl,arylalkyl, heteroaryl, heteroarylalkyl, —NRaRb, —NRaC(═O)Rb,—NRaC(═O)NRaNRb, —NRaC(═O)ORb, —NRaSO2Rb, —C(═O)Ra, —C(═O)ORa,—C(═O)NRaRb, —OC(═O)NRaRb, —ORa, —SRa, —SORa, —S(═O)2Ra, —OS(═O)2Ra and—S(═O)2ORa. Ra and Rb in this context may be the same or different andindependently hydrogen, halogen hydroxy, alkyl, alkoxy, alkyl, amino,alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl,heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl.

The term “optionally substituted,” as used herein, means thatsubstitution is optional and therefore it is possible for the designatedatom to be unsubstituted.

As used herein, “subject” refers to any animal, preferably a humanpatient, livestock, or domestic pet.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, the onset is delayed, or the severity is reduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g. patient) is cured and the disease iseradicated. Rather, embodiments of the present disclosure alsocontemplate treatment that merely reduces symptoms, and/or delaysdisease progression.

As used herein, the term “calcium phosphate(s)” refers to mineralscontaining calcium ions together with orthophosphates, metaphosphates orpyrophosphates and optionally hydroxide ions. Tricalcium phosphate is acalcium phosphate with formula Ca₃(PO₄)₂. The common mineral apatite hasthe basic formula Ca₅(PO₄)₃X, where X is a ion, typically a halogen orhydroxide ion, or a mixture. Hydroxyapatite refers to apatite where X ismainly hydroxide ion. In certain embodiments, the disclosurecontemplates calium phosphates that can further include both silicate(SiO₄ ⁴⁻) and carbonate (CO₃ ²⁻) substituted hydroxyapatites, where thesubstitution is for one or more of the hydroxy and/or phosphate groups.

When used in reference to compound(s) disclosed herein, “salts” refer toderivatives of the disclosed compound(s) where the parent compound ismodified making acid or base salts thereof. Examples of salts include,but are not limited to, mineral or organic acid salts of basic residuessuch as amines, alkylamines, or dialkylamines; alkali or organic saltsof acidic residues such as carboxylic acids; and the like.

As used herein, “alkyl” means a noncyclic straight chain or branched,unsaturated or saturated hydrocarbon such as those containing from 1 to10 carbon atoms. Representative saturated straight chain alkyls includemethyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-septyl, n-octyl,n-nonyl, and the like; while saturated branched alkyls includeisopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.Unsaturated alkyls contain at least one double or triple bond betweenadjacent carbon atoms (referred to as an “alkenyl” or “alkynyl”,respectively). Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like; while representative straightchain and branched alkynyls include acetylenyl, propynyl, 1-butynyl,2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

Non-aromatic mono or polycyclic alkyls are referred to herein as“carbocycles” or “carbocyclyl” groups. Representative saturatedcarbocycles include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,and the like; while unsaturated carbocycles include cyclopentenyl andcyclohexenyl, and the like.

“Heterocarbocycles” or heterocarbocyclyl” groups are carbocycles whichcontain from 1 to 4 heteroatoms independently selected from nitrogen,oxygen and sulfur which may be saturated or unsaturated (but notaromatic), monocyclic or polycyclic, and wherein the nitrogen and sulfurheteroatoms may be optionally oxidized, and the nitrogen heteroatom maybe optionally quaternized. Heterocarbocycles include morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl,oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, and the like.

“Aryl” means an aromatic carbocyclic monocyclic or polycyclic ring suchas phenyl or naphthyl. Polycyclic ring systems may, but are not requiredto, contain one or more non-aromatic rings, as long as one of the ringsis aromatic.

As used herein, “heteroaryl” or “heteroaromatic” refers an aromaticheterocarbocycle having 1 to 4 heteroatoms selected from nitrogen,oxygen and sulfur, and containing at least 1 carbon atom, including bothmono- and polycyclic ring systems. Polycyclic ring systems may, but arenot required to, contain one or more non-aromatic rings, as long as oneof the rings is aromatic. Representative heteroaryls are furyl,benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl,isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl,isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl,thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl,pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl. It iscontemplated that the use of the term “heteroaryl” includes N-alkylatedderivatives such as a 1-methylimidazol-5-yl substituent.

As used herein, “heterocycle” or “heterocyclyl” refers to mono- andpolycyclic ring systems having 1 to 4 heteroatoms selected fromnitrogen, oxygen and sulfur, and containing at least 1 carbon atom. Themono- and polycyclic ring systems may be aromatic, non-aromatic ormixtures of aromatic and non-aromatic rings. Heterocycle includesheterocarbocycles, heteroaryls, and the like.

“Alkylthio” refers to an alkyl group as defined above attached through asulfur bridge. An example of an alkylthio is methylthio, (i.e., —S—CH₃).

“Alkoxy” refers to an alkyl group as defined above attached through anoxygen bridge. Examples of alkoxy include, but are not limited to,methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy,n-pentoxy, and s-pentoxy. Preferred alkoxy groups are methoxy, ethoxy,n-propoxy, propoxy, n-butoxy, s-butoxy, t-butoxy.

“Alkylamino” refers an alkyl group as defined above attached through anamino bridge. An example of an alkylamino is methylamino, (i.e.,—NH—CH₃).

“Alkanoyl” refers to an alkyl as defined above attached through acarbonyl bridge (i.e., —(C═O)alkyl).

“Alkylsulfonyl” refers to an alkyl as defined above attached through asulfonyl bridge (i.e., —S(═O)₂alkyl) such as mesyl and the like, and“Arylsulfonyl” refers to an aryl attached through a sulfonyl bridge(i.e., —S(═O)₂aryl).

“Alkylsulfamoyl” refers to an alkyl as defined above attached through asulfamoyl bridge (i.e., —S(═O)₂NHalkyl), and an “Arylsulfamoyl” refersto an alkyl attached through a sulfamoyl bridge (i.e., —S(═O)₂NHaryl).

“Alkylsulfinyl” refers to an alkyl as defined above with the indicatednumber of carbon atoms attached through a sulfinyl bridge (i.e.—S(═O)alkyl).

The terms “halogen” and “halo” refer to fluorine, chlorine, bromine, andiodine.

The term “bone graft composition” refers to materials that aresubstantially physiologically compatible when residing in bone area,void, or exterior site. In certain embodiments, the bone graftcomposition may be a bone graft matrix such as a collagen sponge or amixture of polymers and salts.

As used herein, the term “retinoic acid” refers to the all-transcompound(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoicacid also known as tretinoin.

Compounds

Compounds derivatives disclosed herein may be used for bone andcartilage growth and related applications. Derivatives of certaincompounds are further exemplified below.

In certain embodiments, the disclosure relates to1-triarylmethyl-1H-imidazole derivatives of formula I:

or a salt thereof, wherein

A, E, and G are each the same or different alkyl, alkynyl, alkenyl, arylor heteroaryl, wherein A, E, and G are optionally substituted with oneor more, the same or different, R⁴;

R¹, R², and R³ are each the same or different hydrogen, alkyl, halogen,nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl,carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl,alkyl sulfonyl, aryl sulfonyl, carbocyclyl, aryl, or heterocyclyl,wherein each R¹, R², and R³ are optionally substituted with one or more,the same or different, R⁴;

R⁴ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkyl sulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R⁴ is optionally substituted with one ormore, the same or different, R⁵; and

R⁵ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl,amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl,methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino,dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino,N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, A is alkynyl.

In certain embodiments, the disclosure relates to1-triarylmethyl-1H-imidazole derivatives of formula IA:

or salts thereof wherein,

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶,R¹⁷ and R¹⁸ are each the same or different hydrogen, alkyl, halogen,nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl,carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl,alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, whereineach R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵,R¹⁶, R¹⁷ and R¹⁸ are optionally substituted with one or more, the sameor different, R¹⁹;

R¹⁹ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkyl sulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R¹⁹ is optionally substituted with one ormore, the same or different, R²⁰; and

R²⁰ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹,R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ are hydrogen and R¹⁸ is a halogen.

In certain embodiments, the disclosure relates to biphenyl-diolderivatives of formula II,

or salts thereof wherein,

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each the same ordifferent hydrogen, alkyl, alkenyl, halogen, nitro, cyano, hydroxy,amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy,alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkyl sulfonyl,aryl sulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R¹, R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰, are optionally substituted with oneor more, the same or different, R¹¹;

R¹¹ is alkyl, alkenyl, halogen, nitro, cyano, hydroxy, amino, mercapto,formyl, carboxy, carbamoyl, alkanoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R¹¹ is optionally substituted with one ormore, the same or different, R¹²; and

R¹² is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R⁴ and R⁹ are alkenyl.

In certain embodiments, the disclosure relates to macrolide lactonederivatives of formula III,

or salts thereof wherein,

the dotted line represents a single or double bond;

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, andR¹⁷ are each the same or different hydrogen, alkyl, alkenyl, halogen,nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl,carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl,alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, whereineach R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵,R¹⁶, and R¹⁷ are optionally substituted with one or more, the same ordifferent, R¹⁸;

R¹⁸ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkyl sulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R¹⁸ is optionally substituted with one ormore, the same or different, R⁹; and

R¹⁹ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R¹ is alkyl or alkenyl wherein R¹ is substitutedwith a carbocyclyl substituted with one or more R¹⁸.

In certain embodiments, R², R³, R⁴, R⁵, R⁷, R⁹, R¹⁰, R¹², and R¹³ areeach the same or different alkyl, alkenyl, hydroxy, or alkoxy.

In certain embodiments, R⁶, R⁸, R¹¹, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ are allhydrogen.

In certain embodiments, the disclosure relates to macrolide lactonederivatives of formula IIIA,

or salts thereof wherein,

the dotted lines each individually represent a single or double bond;

X is hydroxy, amino, mercapto, or halogen;

R², R³, R⁴, R⁵, R⁷, R⁹, R¹⁰, R¹², R¹³, R¹⁸, and R¹⁹ are each the same ordifferent hydrogen, alkyl, alkenyl, halogen, nitro, cyano, hydroxy,amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy,alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R², R³,R⁴, R⁵, R⁷, R⁹, R¹⁰, R¹², R¹³, R¹⁸, and R¹⁹ are optionally substitutedwith one or more, the same or different, R²⁰;

R²⁰ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R²⁰ is optionally substituted with one ormore, the same or different, R²¹; and

R²¹ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R², R³, R⁴, R⁵, R⁷, R⁹, R¹⁰, R¹², R¹³, R¹⁸, andR¹⁹ are each the same or different hydrogen, alkyl, alkenyl, halogen,hydroxy, or alkoxy.

In certain embodiments, X is hydroxy or halogen.

In certain embodiments, R², R⁵, R⁷, and R¹⁸ are alkyl.

In certain embodiments, R³ and R¹³ are hydroxy.

In certain embodiment, R⁴ is alkenyl.

In certain embodiment, R⁹, R¹⁰, and R¹⁹ are alkoxy.

In certain embodiments, the disclosure relates to steroid derivatives.In certain embodiments, the steroid derivatives have formula IV:

or salts thereof wherein,

the dotted lines represent an optional bond to provide an absent,single, or double bond

a) provided that if the dotted ring is aromatic, then the dotted linebetween the O and the ring is a single bond and the dotted line betweenthe ring and R⁵ is absent as R⁵ is absent and

b) provided that if the dotted line between O and the ring is a doublebond, then the dotted line between the O and R⁹ is absent as R⁹ isabsent;

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each the same ordifferent hydrogen, alkyl, alkenyl, alkynyl, halogen, nitro, cyano,hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy,alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R¹, R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are optionally substituted with oneor more, the same or different, R¹¹;

R¹ and R² may form a carbocyclic or heterocyclic ring optionallysubstituted with one or more, the same or different, R¹¹.

R¹¹ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R¹¹ is optionally substituted with one ormore, the same or different, R¹²; and

R¹² is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R¹ and R² form a 5-membered lactone ring.

In certain embodiments, R² is hydroxy.

In certain embodiments, R¹⁰ is hydrogen or mercapto optionallysubstituted with acetyl.

In certain embodiments, the dotted ring is aromatic and R⁹ is hydrogen.

In certain embodiments, the disclosure relates to3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbothioicacid derivatives of formula IVA

or salts thereof wherein,

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are each the same or differenthydrogen, alkyl, alkenyl, halogen, nitro, cyano, hydroxy, amino,mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio,alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,carbocyclyl, aryl, or heterocyclyl, wherein each R¹, R², R³, R⁴, R⁵, R⁶,R⁷, and R⁸ are optionally substituted with one or more, the same ordifferent, R¹⁰,

R¹⁰ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R¹⁰ is optionally substituted with one ormore, the same or different, R¹¹; and

R¹¹ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R¹ is an alkyl substituted with one or morehalogens.

In certain embodiments, R² is hydroxy substituted with an alkanoyl.

In certain embodiments, R⁴ and R⁸ are halogen.

In certain embodiments, R⁶ is hydroxy.

In certain embodiments, the disclosure relates to3-(4-morpholinophenyl)oxazolidin-2-one derivatives of formula V,

or salts thereof wherein

X is O, S, or NR¹⁷

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶,R¹⁷ are each the same or different hydrogen, alkyl, alkenyl, halogen,nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl,carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl,alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, whereineach R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵,R¹⁶, and R¹⁷ are optionally substituted with one or more, the same ordifferent, R¹⁸;

R¹⁸ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R¹⁸ is optionally substituted with one ormore, the same or different, R¹⁹; and

R¹⁹ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, X is O.

In certain embodiments, R¹ is alkyl substituted with R¹⁸.

In certain embodiments, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹²,R¹³, R¹⁴, R¹⁵, and R¹⁶ are all hydrogen.

In certain embodiments, the disclosure relates to3-(4-morpholinophenyl)oxazolidin-2-one derivatives of formula VI,

or salts thereof wherein

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶,R¹⁷ R¹⁸, R¹⁹, and R²⁰ are each the same or different hydrogen, alkyl,halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy,alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, orheterocyclyl, wherein each R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹,R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷ R¹⁸, R¹⁹, and R²⁰ are optionallysubstituted with one or more, the same or different, R²¹;

R²¹ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R²¹ is optionally substituted with one ormore, the same or different, R²²; and

R²² is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R¹, R², and R⁸ are each the same or differentalkyl.

In certain embodiments, R¹⁴ is carboxy.

In certain embodiments, R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁵,R¹⁶, R¹⁷ R¹⁸, R¹⁹, and R²⁰ are hydrogen.

In certain embodiments, the disclosure relates to rentinol derivativesof formula VII,

or salts thereof wherein

the dotted lines represent an optional bond to provide an absent,single, or double bond provided that if the dotted ring is aromatic,then the dotted line between R² and the ring is absent as R² is absent;

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are eachthe same or different hydrogen, alkyl, halogen, nitro, cyano, hydroxy,amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy,alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R¹, R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are optionallysubstituted with one or more, the same or different, R¹⁵;

R¹⁵ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R¹⁵ is optionally substituted with one ormore, the same or different, R¹⁶; and

R¹⁶ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R¹, R², R⁶, R¹¹, and R¹³ are alkyl.

In certain embodiments, R³, R⁴, R⁵, R⁷, R⁸, R⁹, R¹², and R¹⁴ arehydrogen.

In certain embodiments, the dotted ring is aromatic and R¹, R³, R⁴, andR⁶ are alkyl or alkoxy.

In certain embodiments R¹⁰ is hydroxy, formyl, or carboxy optionallysubstituted with R¹⁵.

In certain embodiments, the disclosure relates to rentinol derivativesof formula VIIA,

or salts thereof wherein

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are eachthe same or different hydrogen, alkyl, halogen, nitro, cyano, hydroxy,amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy,alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R¹, R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are optionallysubstituted with one or more, the same or different, R¹⁵;

R¹⁵ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R¹⁵ is optionally substituted with one ormore, the same or different, R¹⁶; and

R¹⁶ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R¹, R², R⁶, R¹¹, and R¹³ are alkyl.

In certain embodiments, R³, R⁴, R⁵, R⁷, R⁸, R⁹, R¹² and R¹⁴ arehydrogen.

In certain embodiments, R¹⁰ is hydroxy, formyl, or carboxy optionallysubstituted with R¹⁵.

In certain embodiments, the disclosure relates to rentinol derivativesof formula VIIB,

or salts thereof wherein

R¹, R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are each thesame or different hydrogen, alkyl, halogen, nitro, cyano, hydroxy,amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy,alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R¹, R³,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are optionallysubstituted with one or more, the same or different, R¹⁵;

R¹⁵ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R¹⁵ is optionally substituted with one ormore, the same or different, R¹⁶; and

R¹⁶ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R¹, R³, R⁶, R¹¹, and R¹³ are alkyl.

In certain embodiments, R⁴ is alkoxy.

In certain embodiments, R⁵, R⁷, R⁸, R⁹, R¹², and R¹⁴ are hydrogen.

In certain embodiments, R¹⁰ is hydroxy, formyl, or carboxy optionallysubstituted with R¹⁵.

In certain embodiments, the disclosure relates to4-(4-(dialkylamino)phenyl)butanoic acid derivatives of formula VIII,

or salts thereof wherein

n is 1, 2, or 3;

m is 1, 2, or 3;

R¹ and R² are each the same or different hydrogen, alkyl, halogen,nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl,carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl,alkylsulfonyl, aryl sulfonyl, carbocyclyl, aryl, or heterocyclyl,wherein each R¹ and R² are optionally substituted with one or more, thesame or different, R¹⁴;

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and R¹³ are each the same ordifferent hydrogen, alkyl, halogen, nitro, cyano, hydroxy, amino,mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio,alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,carbocyclyl, aryl, or heterocyclyl, wherein each R³, R⁴, R⁵, R⁶, R⁷, R⁸,R⁹, R¹⁰, R¹¹, R¹², and R¹³ are optionally substituted with one or more,the same or different, R¹⁴;

R¹⁴ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R¹⁴ is optionally substituted with one ormore, the same or different, R¹⁵; and

R¹⁵ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R¹ and R² are a halogen.

In certain embodiments, R³, R⁴, R⁵, and R⁶ are hydrogen.

In certain embodiments, R⁷, R⁸, R⁹, R¹⁰, R¹¹, and R¹² are hydrogen.

In certain embodiments, R¹³ is hydrogen or alkyl.

In certain embodiments, the disclosure relates to macrolide lactonederivatives of formula IX,

or salts thereof wherein,

X is hydroxy, amino, mercapto, halogen, or —O(CH₂)_(n)OH wherein X isoptionally substituted with one or more, the same or different, R²⁰;

n is 2, 3, or 4;

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁸, and R¹⁹ are eachthe same or different hydrogen, alkyl, alkenyl, halogen, nitro, cyano,hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy,alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R¹, R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁸, and R¹⁹ are optionallysubstituted with one or more, the same or different, R²⁰;

R²⁰ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R²⁰ is optionally substituted with one ormore, the same or different, R²¹; and

R²¹ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹²,R¹³, R¹⁸, and R¹⁹ are each the same or different hydrogen, alkyl,halogen, hydroxy, or alkoxy.

In certain embodiments, X is hydroxy or —O(CH₂)_(n)OH.

In certain embodiments, n is 2.

In certain embodiments, R¹, R², R⁵, R⁶, R⁷, R¹², and R¹⁸ are alkyl.

In certain embodiments, R³ and R¹³ are hydroxy.

In certain embodiment, R⁴, R⁸, and R¹⁹ are alkoxy.

In certain embodiments, the podophyllotoxin derivative is a compound offormula X,

or salts thereof wherein,

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each the same ordifferent hydrogen, alkyl, alkenyl, halogen, nitro, cyano, hydroxy,amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy,alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R¹, R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are optionally substituted with oneor more, the same or different, R¹¹;

R¹¹ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R¹¹ is optionally substituted with one ormore, the same or different, R¹²; and

R¹² is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R⁵, R⁸, R⁹, and R¹⁰ are hydrogen.

In certain embodiments, R², R³, R⁴, R⁵, R⁶, and R⁷ are each are each thesame or different hydroxy or alkoxy.

In certain embodiments, the aziridine is derivative is a compound offormula XI,

or salts thereof wherein,

R¹, R², R³, R⁴, and R⁵ are each the same or different hydrogen, alkyl,alkenyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein each R¹, R², R³, R⁴, and R⁵ areoptionally substituted with one or more, the same or different, R⁶;

R⁶ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R⁶ is optionally substituted with one ormore, the same or different, R⁷; and

R⁷ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl,amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl,methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino,dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino,N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R¹ is alkyl.

In certain embodiments, R² is amino.

In certain embodiments, R³ is alkyl substituted with a carbamoyl group.

In certain embodiments, R⁴ is alkoxy.

In certain embodiments, the nucleoside derivative is a compound offormula XII,

or salts thereof wherein,

X is N or CR⁶;

R¹, R², R³, R⁴, R⁵, and R⁶ are each the same or different hydrogen,alkyl, alkenyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein each R¹, R², R³, R⁴, R⁵, and R⁶ areoptionally substituted with one or more, the same or different, R⁷;

R⁷ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R⁷ is optionally substituted with one ormore, the same or different, R⁸; and

R⁸ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl,amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl,methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino,dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino,N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R¹ is hydroxymethyl.

In certain embodiments, R² is hydroxy.

In certain embodiments, R³ is hydrogen or hydroxy.

In certain embodiments, R⁴ is hydrogen.

In certain embodiments, R⁵ is hydrogen or amino.

In certain embodiments, X is CH or N.

In certain embodiments, the vinca alkaloid derivative is a compound offormula XIII,

or salts thereof wherein,

n is 1 or 2;

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶,R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, and R²⁵ are each the same ordifferent hydrogen, alkyl, alkenyl, halogen, nitro, cyano, hydroxy,amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy,alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R¹, R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸,R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, and R²⁵ are optionally substituted withone or more, the same or different, R²⁶;

R²⁶ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R²⁶ is optionally substituted with one ormore, the same or different, R²⁷; and

R²⁷ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R⁴ is alkyl.

In certain embodiments, R⁵ is hydroxy substituted with acetyl.

In certain embodiments, R⁷ is hydroxy.

In certain embodiments, R⁸ is carboxy substituted with alkyl.

In certain embodiments, R¹⁰ is alkyl or formyl.

In certain embodiments, R¹² is alkoxy.

In certain embodiments, R¹³ is carboxy substituted with alkyl.

In certain embodiments, R¹⁶ and R¹⁸ form a double bond.

In certain embodiments, R¹⁸ is hydroxy.

In certain embodiments, R¹⁹ is alkyl.

In certain embodiments, R¹, R², R³, R⁶, R⁹, R¹¹, R¹⁴, R¹⁵, R¹⁶, R¹⁷,R²⁰, R²¹, R²², R²³, R²⁴, and R²⁵ are each hydrogen.

In certain embodiments, the anthracene-9,10-dione derivatives is acompound of formula XIV,

or salts thereof wherein,

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are each the same or differenthydrogen, alkyl, alkenyl, halogen, nitro, cyano, hydroxy, amino,mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio,alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,carbocyclyl, aryl, or heterocyclyl, wherein each R², R³, R⁴, R⁵, R⁶, R⁷,and R⁸ are optionally substituted with one or more, the same ordifferent, R⁹;

R⁹ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R⁹ is optionally substituted with one ormore, the same or different, R¹⁰; and

R¹⁰ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, the anthracycline derivatives is a compound offormula XV,

or salts thereof wherein,

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are eachthe same or different hydrogen, alkyl, alkenyl, halogen, nitro, cyano,hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy,alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R², R³,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are optionallysubstituted with one or more, the same or different, R¹⁵;

R¹⁵ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R¹⁵ is optionally substituted with one ormore, the same or different, R¹⁶; and

R¹⁶ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R¹ and R¹⁰ are hydroxy.

In certain embodiments, R¹¹ is alkoxy.

In certain embodiments, R⁴ is hydroxy.

In certain embodiments, R⁵ is alkanoyl.

In certain embodiments, R⁸ is (oxan-2-yl)oxy or(4-amino-5-hydroxy-6-methyl-oxan-2-yl)oxy.

In certain embodiments, the pazopanib derivatives are compounds offormula XVI,

or salts thereof wherein,

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², and R¹³ are each thesame or different hydrogen, alkyl, alkenyl, halogen, nitro, cyano,hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy,alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R¹, R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², and R¹³ are optionallysubstituted with one or more, the same or different, R¹⁴;

R¹⁴ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R¹⁴ is optionally substituted with one ormore, the same or different, R¹⁵; and

R¹⁵ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, the camptothecin derivative is a compound of theformula XVII,

or salts thereof wherein,

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, and R¹² are each the sameor different hydrogen, alkyl, alkenyl, halogen, nitro, cyano, hydroxy,amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy,alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R¹, R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, and R¹² are optionally substitutedwith one or more, the same or different, R¹³;

R¹³ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R¹³ is optionally substituted with one ormore, the same or different, R¹⁴; and

R¹⁴ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, the sunitinib derivatives have formula XVII,

or salts thereof wherein,

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ are each the same ordifferent hydrogen, alkyl, alkenyl, halogen, nitro, cyano, hydroxy,amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy,alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R¹, R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ are optionally substituted withone or more, the same or different, R¹²;

R¹² is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl,aryl, or heterocyclyl, wherein R¹² is optionally substituted with one ormore, the same or different, R¹³; and

R¹³ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

Evaluations of Compound Activity

BMP-2 and BMP-4 have been established to be important factors inembryonic skeletal development. BMP receptors are transmembranereceptors classified as type I or type II based on sequence homology andcontain a Ser/Thr protein kinase domain. BMP ligand binding to type Ireceptor (BMPR-I) induces the association of BMPR-I and BMPR-IIreceptors, allowing the constitutively phosphorylated BMPR-II tophosphorylate and activate the latent BMPR-I. After activation ofBMPR-I, the receptor regulated (R)-Smad1/5/8 is phosphorylated.Phosphorylation of R-Smad releases it from the receptor complex andforms hetero-complex associating with common Smad (Co-Smad, Smad4).Subsequently, R-Smad/Co-Smad complex translocates into the nucleus andregulates the transcription of target genes by functioning in concertwith other proteins as transcription factors. See FIG. 1. BMP activityis regulated prior to receptor recognition by the presence of severalstructurally distinct extracellular BMP antagonists such as Noggin,follistatin, sclerostatin, chordin, DCR, BMPMER, cerberus, gremlin, DAN,and others.

Structures of the BMPs and its receptors were analyzed to identify thevarious residues involved in their interaction. Using the LUDI de novodesign method a large number of compounds were computationally screenedagainst the binding sites of BMP-receptors to identify lead chemicalcompounds that mimic BMP-receptor interactions. Small molecules wereidentified that bind to BMP-receptor binding epitopes or facilitate BMPbinding to the receptor or block noggin binding to BMP. Their activitywas evaluated by determining the potentiation of alkaline phosphataseactivity in sub-optimal doses of BMP in the presence or absence ofexogenously added noggin.

Data summary for selected compounds is provided in the table below:

Fold increase Fold increase in BMP-Potentiated in Noggin-Inhibition ALPactivity over ALP Assay over Compound BMP control BMP alone controlClotrimazole 7 4 Honokiol 5 16 Tacrolimus 7 5 Fluticasone Propionate 6 3Linezolid 5 3 Telmisartan 3 3 Retinoic acid 11 2 Spironolactone 3 4Chlorambucil 3 4 Isotretinoin 30 3 Acitretin 33 3

The fold-increase in Noggin-inhibition assay is based on BMP-alonecontrol. Further 3 to 5-fold increases were observed when BMP and Noggincontrol is compared.

Data for additions compounds is provided below.

Fold-increase of BMP-induced ALP Compound (over BMP alone control)Teniposide 7.0 Mitomycin C 12.0 Cytarabine HCl 2.0 Vinorelbine tartrate4.0 Mitoxantrone 4.0 Plicamycin 7.0 Vincristine 5.0 Valrubicin 3.0Doxorubicin 4.0 Pazopanib 2.5 Topotecan 4.0 Decitabine 6.0 Sunitinib 4.0Daunomycin, monohydrochloride 7.0 9-aminocamptothecin 10.01-(1,1-diphenylprop- 7.0 2-yn-1-yl)-1H-imidazole

In certain embodiments, the disclosure contemplates compositionscomprising any of the the compounds herein, or derivatives orsubstituted forms, such as those in the table, and uses for any of theapplications disclosed herein.

Growth Factors

In some embodiments, the disclosure relates to the combined use ofgrowth factor(s) and compounds disclosed herein such as a compoundselected from clotrimazole, honokiol, magnolol, tacrolimus,pimecrolimus, sirolimus, everolimus, temsirolimus, spironolactone,fluticasone, fluticasone propionate, fluticasone furoate, linezolid,telmisartan, chlorambucil, retinol, isotretinoin, acitretin, etretinate,retinoic acid (tretinoin), teniposide, mitomycin C, cytarabine,decitabine, vinblastine, vincristine, vindesine, vinorelbine,valrubicin, doxorubicin, daunorubicin, epirubicin, idarubicin,mitoxantrone, pixantrone, plicamycin, pazopanib, topotecan,camptothecin, irinotecan, sunitinib, derivatives, or salt thereof andone or more growth factors in bone growth applications. Typically, thegrowth factor is a bone morphogenetic proteins (BMPs), including but notlimited to, BMP-1, BMP-2, BMP-2A, BMP-2B, BMP-3, BMP-3b, BMP-4, BMP-5,BMP-6, BMP-7 (OP-1), BMP-8, BMP-8b, BMP-9, BMP-10, BMP-11, BMP-12,BMP-13, BMP-14, BMP-15. BMPs act through specific transmembranereceptors located on cell surface of the target cells.

Non-limiting examples of additional suitable growth factors includeosteogenin, insulin-like growth factor (IGF)-1, IGF-II, TGF-beta1,TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, osteoinductive factor (OIF),basic fibroblast growth factor (bFGF), acidic fibroblast growth factor(aFGF), platelet-derived growth factor (PDGF), vascular endothelialgrowth factor (VEGF), growth hormone (GH), growth and differentiationfactors (GDF)-5 through 9, and osteogenic protein-1 (OP-1). The growthfactors may be isolated from synthetic methods, recombinant sources ormay be purified from a biological sample. Preferably the growth factorsare obtained from a recombinant technology and for clarity certainembodiments include rhBMP-2, rhBMP-4, rhBMP-6, rhBMP-7, and rhGDF-5, asdisclosed, for example, in the U.S. Pat. Nos. 4,877,864; 5,013,649;5,661,007; 5,688,678; 6,177,406; 6,432,919; 6,534,268, and 6,858,431,and in Wozney, J. M., et al. (1988) Science, 242(4885):1528-1534 herebyincorporated by reference.

In a typical embodiment, a graft composition comprises a matrix, BMP-2,and one or more compound(s) disclosed herein such a compound selectedfrom clotrimazole, honokiol, magnolol, tacrolimus, pimecrolimus,sirolimus, everolimus, temsirolimus, spironolactone, fluticasone,fluticasone propionate, fluticasone furoate, linezolid, telmisartan,chlorambucil, retinol, isotretinoin, acitretin, etretinate, retinoicacid (tretinoin), teniposide, mitomycin C, cytarabine, decitabine,vinblastine, vincristine, vindesine, vinorelbine, valrubicin,doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone,pixantrone, plicamycin, pazopanib, topotecan, camptothecin, irinotecan,sunitinib, derivatives, or salt thereof in combinations with othergrowth factors such as GDF-5. In one embodiment, the matrix contains aneffective amount of a BMP-2 protein, an rhBMP-2 protein, functionalfragments thereof, or combinations thereof. For certain embodiments, therange of concentrations of BMP-2 may be about 1.0 to 4.0 mg/ml and GDF-5concentrations may be 0.25 to 4.0 mg/ml. Although a graft matrix may beloaded during manufacturing, it is typically loaded just prior toimplantation.

The polypeptide of human BMP-2 is 396 amino acids in length, and itsgene is localized to chromosome 20p12. BMP-2 belongs to the transforminggrowth factor-beta (TGF-beta) superfamily. The human amino acid sequenceBMP-2 is SEQ ID NO:1 shown below. Amino acids 38-268 are the TGF-betapropeptide domain, and 291-396 are the TGF-beta family N-terminaldomain. Amino acids 283-396 are the mature peptide. The mature form ofBMP-2 contains four potential N-linked glycosylation sites perpolypeptide chain, and four potential disulfide bridges. (SEQ ID NO: 1)1 MVAGTRCLLA LLLPQVLLGG AAGLVPELGR RKFAAASSGR PSSQPSDEVL SEFELRLLSM 61FGLKQRPTPS RDAVVPPYML DLYRRHSGQP GSPAPDHRLE RAASRANTVR SFHHEESLEE 121LPETSGKTTR RFFFNLSSIP TEEFITSAEL QVFREQMQDA LGNNSSFHHR INIYEIIKPA 181TANSKFPVTR LLDTRLVNQN ASRWESFDVT PAVMRWTAQG HANHGFVVEV AHLEEKQGVS 241KRHVRISRSL HQDEHSWSQI RPLLVTFGHD GKGHPLHKRE KRQAKHKQRK RLKSSCKRHP 301LYVDFSDVGW NDWIVAPPGY HAFYCHGECP FPLADHLNST NHAIVQTLVN SVNSKIPKAC 361CVPTELSAIS MLYLDENEKV VLKNYQDMVV EGCGCR.

In one embodiment, bone morphogenetic protein includes one of thefollowing synthetic peptide fragments of BMP-2: SEQ ID NO: 2(KIPKASSVPTELSAISTLYLDDD), SEQ ID NO: 3 (CCCCDDDSKIPKASSVPTELSAISTLYL)SEQ ID NO: 4 (C₁₆H₃₁O—NH-CCCCGGGSKIPKASSVPTELSAISTLYL) which may besynthesized by FMOC/tBu solid-phase peptide synthesis.

BMP-7 also belongs to the TGF-beta superfamily. It induces cartilage andbone formation. The amino acid sequence of BMP-7 is SEQ ID NO: 5. (SEQID NO: 5) 1 MHVRSLRAAA PHSFVALWAP LFLLRSALAD FSLDNEVHSS FIHRRLRSQERREMQREILS 61 ILGLPHRPRP HLQGKHNSAP MFMLDLYNAM AVEEGGGPGG QGFSYPYKAVFSTQGPPLAS 121 LQDSHFLTDA DMVMSFVNLV EHDKEFFHPR YHHREFRFDL SKIPEGEAVTAAEFRIYKDY 181 IRERFDNETF RISVYQVLQE HLGRESDLFL LDSRTLWASE EGWLVFDITATSNHWVVNPR 241 HNLGLQLSVE TLDGQSINPK LAGLIGRHGP QNKQPFMVAF FKATEVHFRSIRSTGSKQRS 301 QNRSKTPKNQ EALRMANVAE NSSSDQRQAC KKHELYVSFR DLGWQDWIIAPEGYAAYYCE 361 GECAFPLNSY MNATNHAIVQ TLVHFINPET VPKPCCAPTQ LNAISVLYFDDSSNVILKKY 421 RNNVVRACGC H. Amino acids 1-29 are a potential signalsequence; 30-431 are the prepropeptide, and 293-431 are the matureprotein. The mature form of BMP-7 contains four potential N-linkedglycosylation sites per polypeptide chain, and four potential disulfidebridges.

Graft Compositions

In some embodiments, the disclosure relates to a graft compositioncomprising growth factor(s) and clotrimazole, honokiol, magnolol,tacrolimus, pimecrolimus, sirolimus, everolimus, temsirolimus,spironolactone, fluticasone, fluticasone propionate, fluticasonefuroate, linezolid, telmisartan, chlorambucil, retinol, isotretinoin,acitretin, etretinate, retinoic acid (tretinoin), teniposide, mitomycinC, cytarabine, decitabine, vinblastine, vincristine, vindesine,vinorelbine, valrubicin, doxorubicin, daunorubicin, epirubicin,idarubicin, mitoxantrone, pixantrone, plicamycin, pazopanib, topotecan,camptothecin, irinotecan, sunitinib, derivatives, or salts thereof. Insome embodiments, these compositions can be created from polymers, bonegranules, and ceramics such as calcium phosphates (e.g. hydroxyapatiteand tricalcium phosphate), bioglass, and calcium sulphate.

Bioglass refers to materials of SiO₂, Na₂O, CaO, and P₂O₅ in specificproportions. The proportions differ from the traditional soda-limeglasses in lower amounts of silica (typically less than 60 mol %),higher amounts of sodium and calcium, and higher calcium/phosphorusratio. A high ratio of calcium to phosphorus promotes formation ofapatite crystals; calcium and silica ions can act as crystallizationnuclei. Some formulations bind to soft tissues and bone, some only tobone, some do not form a bond at all and after implantation getencapsulated with non-adhering fibrous tissue, and others are completelyabsorbed overtime. Mixtures of 35-60 mol % SiO₂, 10-50 mol % CaO, and5-40 mol % Na₂O bond to bone and some formulations bond to soft tissues.Mixtures of >50 mol % SiO₂, <10 mol % CaO, <35 mol % Na₂O typicallyintegrate within a month. Some CaO may be replaced with MgO and someNa₂O may be replaced with K₂O. Some CaO can be replaced with CaF₂.

In some embodiments, the disclosure relates to a graft compositioncomprising growth factor(s) and compounds disclosed herein such asclotrimazole, honokiol, magnolol, tacrolimus, pimecrolimus, sirolimus,everolimus, temsirolimus, spironolactone, fluticasone, fluticasonepropionate, fluticasone furoate, linezolid, telmisartan, chlorambucil,retinol, isotretinoin, acitretin, etretinate, retinoic acid (tretinoin),teniposide, mitomycin C, cytarabine, decitabine, vinblastine,vincristine, vindesine, vinorelbine, valrubicin, doxorubicin,daunorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone,plicamycin, pazopanib, topotecan, camptothecin, irinotecan, sunitinib,derivatives, or salts thereof and/or polysaccharides such ashyaluronate, cellulose or cellulose derivatives such as, but not limitedto, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, andcarboxymethyl cellulose. Typically, cellulose derivatives are used ingraft compositions that produce a paste or putty.

In some embodiments, the disclosure relates to a bone graft compositioncomprising a bone morphogenetic protein and clotrimazole, honokiol,magnolol, tacrolimus, pimecrolimus, sirolimus, everolimus, temsirolimus,spironolactone, fluticasone, fluticasone propionate, fluticasonefuroate, linezolid, telmisartan, chlorambucil, retinol, isotretinoin,acitretin, etretinate, retinoic acid (tretinoin), teniposide, mitomycinC, cytarabine, decitabine, vinblastine, vincristine, vindesine,vinorelbine, valrubicin, doxorubicin, daunorubicin, epirubicin,idarubicin, mitoxantrone, pixantrone, plicamycin, pazopanib, topotecan,camptothecin, irinotecan, sunitinib, derivatives, or salt thereof and agraft matrix. The matrix is typically a polymer designed to hold bonecompatible salts, such as calcium phosphates, for replacement duringbone growth. An example is a bovine Type I collagen embedded withbiphasic calcium phosphate granules. Optionally, matrix compositions mayalso include one or more agents that support the formation, developmentand growth of new bone, and/or the remodeling thereof. Typical examplesof compounds that function in, such a supportive manner includeextracellular matrix-associated bone proteins such as alkalinephosphatase, osteocalcin, bone sialoprotein (BSP) and osteocalcin,phosphoprotein (SPP)-1, type I collagen, fibronectin, osteonectin,thrombospondin, matrix-gla-protein, SPARC, and osteopontin.

In certain embodiments, the graft matrix can be made up of a hydrogelpolymer. Typically, a hydrogel is made-up of acrylate polymers andcopolymers substituted with an abundance of hydrophilic groups, such asterminal hydroxy or carboxyl groups. In certain embodiments, the graftcomposition is biodegradable. In certain embodiments, the matrixcomprises homopolymers and copolymers consisting of gylcolide andlactide. For certain embodiments, the graft composition comprises amatrix of hydroxyethylmethacrylate or hydroxymethylmethyacrylatepolymers containing hydroxyapatite in a mineral content approximatelythat of human bone. Such a composition may also be made withcrosslinkers comprising an ester, anhydride, orthoester, amide, orpeptide bond. In some embodiments, crosslinkers contain the followingpolymers: polyethylene glycol (PEG), polylactic acid, polyglycolide orcombinations thereof.

In certain embodiments, the graft can be any material that is porousincluding those that have macroporosity (mean pore diameter greater thanor equal to 100 μm), mesoporosity (mean pore diameter less than 100 μmbut greater than or equal to 10 μm) and microporosity (mean porediameter less than 10 μm). The pores may be of any size, shape ordistribution, or within a predetermined tolerance. In addition, thepores can be interconnecting or non-interconnecting. In certainembodiments, the graft composition can be a plurality of porous ornon-porous granules. The specific surface area of the graft can vary.For example, when the graft is a porous granule, the specific surfacearea can range from about 0.1 m²/g to about 100 m²/g.

Suitable polymers useful for preparing the graft include, but are notlimited to, homopolymers or copolymers of monomers selected fromL-lactide; L-lactic acid; D-lactide; D-lactic acid; glycolide;alpha-hydroxybutyric acid; alpha-hydroxyvaleric acid;alpha-hydroxyacetic acid; alpha-hydroxycaproic acid;alpha-hydroxyheptanoic acid; alpha-hydroxydecanoic acid;alpha-hydroxymyristic acid; alpha-hydroxyoctanoic acid;alpha-hydroxystearic acid; hydroxybutyrate; hydroxyvalerate;beta-propiolactide; beta-propiolactic acid; gamma-caprolactone;beta-caprolactone; epsilon-caprolactone; gamma-butyrolactone;pivalolactone; tetramethylglycolide; tetramethylglycolic acid;dimethylglycolic acid; trimethylene carbonate; dioxanone; those monomersthat form liquid crystal polymers; those monomers that form cellulose;those monomers that form cellulose acetate; those monomers that formcarboxymethylcellulose; those monomers that formhydroxypropylmethylcellulose; polyurethane precursors includingmacrodiols selected from polycaprolactone, poly(ethylene oxide),poly(ethylene glycol), poly(ethylene adipate), poly(butylene oxide), anda mixture thereof, isocyanate-functional compounds selected fromhexamethylene diisocyanate, isophorone diisocyanate, cyclohexanediisocyanate, hydrogenated methylene diphenylene diisocyanate, and amixture thereof, and chain extenders selected from ethylenediamine,1,4-butanediol, 1,2-butanediol, 2-amino-1-butanol, thiodiethylene diol,2-mercaptoethyl ether, 3-hexyne-2,5-diol, citric acid, and a mixturethereof, and any combination of two or more of the foregoing.

In certain embodiments, the graft composition may contain one or moreantibiotics and/or anti-inflammatory agents. Suitable antibioticsinclude, without limitation, nitroimidazole antibiotics, tetracyclines,penicillins, cephalosporins, carbopenems, aminoglycosides, macrolideantibiotics, lincosamide antibiotics, 4-quinolones, rifamycins andnitrofurantoin. Suitable specific compounds include, without limitation,ampicillin, amoxicillin, benzylpenicillin, phenoxymethylpenicillin,bacampicillin, pivampicillin, carbenicillin, cloxacillin, cyclacillin,dicloxacillin, methicillin, oxacillin, piperacillin, ticarcillin,flucloxacillin, cefuroxime, cefetamet, cefetrame, cefixine, cefoxitin,ceftazidime, ceftizoxime, latamoxef, cefoperazone, ceftriaxone,cefsulodin, cefotaxime, cephalexin, cefaclor, cefadroxil, cefalothin,cefazolin, cefpodoxime, ceftibuten, aztreonam, tigemonam, erythromycin,dirithromycin, roxithromycin, azithromycin, clarithromycin, clindamycin,paldimycin, lincomycirl, vancomycin, spectinomycin, tobramycin,paromomycin, metronidazole, tinidazole, ornidazole, amifloxacin,cinoxacin, ciprofloxacin, difloxacin, enoxacin, fleroxacin, norfloxacin,ofloxacin, temafloxacin, doxycycline, minocycline, tetracycline,chlortetracycline, oxytetracycline, methacycline, rolitetracyclin,nitrofurantoin, nalidixic acid, gentamicin, rifampicin, amikacin,netilmicin, imipenem, cilastatin, chloramphenicol, furazolidone,nifuroxazide, sulfadiazin, sulfametoxazol, bismuth subsalicylate,colloidal bismuth subcitrate, gramicidin, mecillinam, cloxiquine,chlorhexidine, dichlorobenzylalcohol, methyl-2-pentylphenol or anycombination thereof.

Suitable anti-inflammatory compounds include both steroidal andnon-steroidal structures. Suitable non-limiting examples of steroidalanti-inflammatory compounds are corticosteroids such as hydrocortisone,cortisol, hydroxyltriamcinolone, alpha-methyl dexamethasone,dexamethasone-phosphate, beclomethasone dipropionates, clobetasolvalerate, desonide, desoxymethasone, desoxycorticosterone acetate,dexamethasone, dichlorisone, diflorasone diacetate, diflucortolonevalerate, fluadrenolone, fluclorolone acetonide, fludrocortisone,flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortinebutylesters, fluocortolone, fluprednidene (fluprednylidene) acetate,flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisonebutyrate, methylprednisolone, triamcinolone acetonide, cortisone,cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate,fluradrenolone, fludrocortisone, diflurosone diacetate, fluocinolone,fluradrenolone acetonide, medrysone, amcinafel, amcinafide,betamethasone and the balance of its esters, chloroprednisone,chlorprednisone acetate, clocortelone, clescinolone, dichlorisone,diflurprednate, flucloronide, flunisolide, fluoromethalone, fluperolone,fluprednisolone, hydrocortisone valerate, hydrocortisonecyclopentylpropionate, hydrocortamate, meprednisone, paramethasone,prednisolone, prednisone, beclomethasone dipropionate, triamcinolone.Mixtures of the above steroidal anti-inflammatory compounds may also beused.

Non-limiting examples of non-steroidal anti-inflammatory compoundsinclude nabumetone, celecoxib, etodolac, nimesulide, apasone, gold,oxicams, such as piroxicam, isoxicam, meloxicam, tenoxicam, sudoxicam,the salicylates, such as aspirin, disalcid, benorylate, trilisate,safapryn, solprin, diflunisal, and fendosal; the acetic acidderivatives, such as diclofenac, fenclofenac, indomethacin, sulindac,tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acematacin,fentiazac, zomepirac, clindanac, oxepinac, felbinac, and ketorolac; thefenamates, such as mefenamic, meclofenamic, flufenamic, niflumic, andtolfenamic acids; the propionic acid derivatives, such as ibuprofen,naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen,indopropfen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen,tioxaprofen, suprofen, alminoprofen, and tiaprofenic; and the pyrazoles,such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone, andtrimethazone.

In certain embodiments, compounds disclosed herein can be combined withthe graft preoperatively as well as intra-operatively. Where thecompound is combined pre-operatively, it can be combined with the graftas part of a manufacturing process where the compound could be appliedto the scaffold in a buffered solution and then subsequently lyophilizedor air dried. The compound may also be applied by spray drying or othercoating methods. The graft could then be subsequently packaged andsterilized. Where the compound is combined intra-operatively with thegraft, the graft can be dipped or coated with a buffered solutionincluding the compound and then applied to the bone site to be repaired.

In certain embodiments, the graft can further include an osteogenicmaterial to provide a viable cell population to the bone repair site.The osteogenic material can be obtained from both autogenic sources aswell as allogenic sources, such as cadaveric sources or tissue banksSuitable osteogenic material can include, for example, viable cellsources such as stem cells, multipotent cells, pluripotent cells,osteoprogenitor cells, pre-osteoblasts, mature osteoblasts, and blendsand mixtures thereof.

In certain embodiments, the osteogenic material is obtained fromautogenic and/or allogenic human bone marrow, and according to anotherembodiment, the osteogenic material is obtained from autogenic and/orallogenic human lipoaspirate. Both the bone marrow and lipoaspirate canbe processed to further enhance the desired cell population for exampleby filtration, separation and/or concentration. In order to preserve theviability of the cell population of the osteogenic material, theosteogenic material is typically combined with the graft matrix andosteoinductive material at or near the time of the implantationprocedure.

Methods

Bone grafting is possible because bone tissue, unlike most othertissues, has the ability to regenerate if provided the space into whichto grow with appropriate chemical signals. With regard to syntheticgrafts, as native bone grows, it typically replaces most or all of theartificial graft material, resulting in an integrated region of newbone. However, with regard to certain embodiments of the disclosure, itis not intended that new bone must remove all artificial material. Inaddition, with regard to certain embodiments of the disclosure, it isnot intended that graft location need contact any other bone of theskeletal system.

In certain embodiments, the disclosure relates to a method of formingbone comprising implanting a graft composition comprising a compounddisclosed herein such as clotrimazole, honokiol, magnolol, tacrolimus,pimecrolimus, sirolimus, everolimus, temsirolimus, spironolactone,fluticasone, fluticasone propionate, fluticasone furoate, linezolid,telmisartan, chlorambucil, retinol, isotretinoin, acitretin, etretinate,retinoic acid (tretinoin), teniposide, mitomycin C, cytarabine,decitabine, vinblastine, vincristine, vindesine, vinorelbine,valrubicin, doxorubicin, daunorubicin, epirubicin, idarubicin,mitoxantrone, pixantrone, plicamycin, pazopanib, topotecan,camptothecin, irinotecan, sunitinib, derivatives, or salts thereof, in asubject. In certain embodiments, the disclosure relates to methods offorming bone comprising implanting a graft composition comprising a bonemorphogenetic protein and compound(s) disclosed herein, such asclotrimazole, honokiol, magnolol, tacrolimus, pimecrolimus, sirolimus,everolimus, temsirolimus, spironolactone, fluticasone, fluticasonepropionate, fluticasone furoate, linezolid, telmisartan, chlorambucil,retinol, isotretinoin, acitretin, etretinate, retinoic acid (tretinoin),teniposide, mitomycin C, cytarabine, decitabine, vinblastine,vincristine, vindesine, vinorelbine, valrubicin, doxorubicin,daunorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone,plicamycin, pazopanib, topotecan, camptothecin, irinotecan, sunitinib,derivatives, or salts thereof, in a subject. The graft may be the resultof a void created by surgical removal or created as a result of anattempt to correct a physical abnormality of a bone, such as but notlimited to, cranial bones; frontal, parietal, temporal, occipital,sphenoid, ethmoid; facial bones; mandible, maxilla, palatine, zygomatic,nasal, lacrimal, vomer, inferior nasal conchae; shoulder girdle; scapulaor shoulder blade, clavicle or collarbone; in the thorax; sternum,manubrium, gladiolus, and xiphoid process, ribs; in the vertebralcolumn; cervical vertebrae, thoracic vertebrae; lumbar vertebrae; in thearms, humerus, radius, ulna; in the pelvis; coccyx; sacrum, hip bone(innominate bone or coxal bone); in the legs; femur, patella, tibia, andfibula. It is contemplated that the graft may be added for cosmeticpurposes, e.g., cheek augmentation. In the case of a broken bone orremoval of a bone during surgery, it may be desirable to secure movementof bone structure with a fixation system and remove the system afterbone forms in the implanted graft.

With regard to prostheses, it may be desirable to grow bone betweenexisting bone and an implanted device, or in preparation of an implanteddevice, such as in the case of a hip replacement, knee replacement, anddental implant, i.e., artificial tooth root used to support restorationsthat resemble a tooth or group of teeth.

In some embodiments, the disclosure relates to three-dimensionalstructures made of biocompatible and biodegradable bone graft materialsin the shape of the bone infused with compositions disclosed herein topromote bone growth. Implants can be used to support a number ofprostheses. A typical implant consists of a titanium device. In certainembodiments, the graft compositions disclosed herein contain implants.

With regard to a sinus augmentation or alveolar ridge augmentation,surgery may be performed as an outpatient under general anesthesia, oralconscious sedation, nitrous oxide sedation, intravenous sedation orunder local anesthesia. Bone grafting is used in cases where there is alack of adequate maxillary or mandibular bone in terms of depth orthickness. Sufficient bone is needed in three dimensions to securelyintegrate with the root-like implant. Improved bone height is importantto assure ample anchorage of the root-like shape of the implant.

In a typical procedure, the clinician creates a large flap of thegingiva or gum to fully expose the bone at the graft site, performs oneor several types of block and onlay grafts in and on existing bone, theninstalls a membrane designed to repel unwanted infection-causingbacteria. Then the mucosa is carefully sutured over the site. Togetherwith a course of systemic antibiotics and topical antibacterial mouthrinses, the graft site is allowed to heal. The bone graft produces livevascular bone and is therefore suitable as a foundation for the dentalimplants.

In certain embodiments, the disclosure relates to methods of performingspinal fusion using compositions disclosed herein. Typically thisprocedure is used to eliminate the pain caused by abnormal motion of thevertebrae by immobilizing the vertebrae themselves. Spinal fusion isoften done in the lumbar region of the spine, but the term is notintended to be limited to method of fusing lumbar vertebrae. Patientsdesiring spinal fusion may have neurological deficits or severe painwhich has not responded to conservative treatment. Conditions wherespinal fusion may be considered include, but are not limited to,degenerative disc disease, spinal disc herniation, discogenic pain,spinal tumor, vertebral fracture, scoliosis, kyphosis (i.e,Scheuermann's disease), spondylolisthesis, or spondylosis.

In certain embodiments, different methods of lumbar spinal fusion may beused in conjunction with each other. In one method, one places the bonegraft between the transverse processes in the back of the spine. Thesevertebrae are fixed in place with screws and/or wire through thepedicles of each vertebra attaching to a metal rod on each side of thevertebrae. In another method, one places the bone graft between thevertebra in the area usually occupied by the intervertebral disc. Inpreparation for the spinal fusion, the disc is removed entirely. Adevice may be placed between the vertebrae to maintain spine alignmentand disc height. The intervertebral device may be made from eitherplastic or titanium or other suitable material. The fusion then occursbetween the endplates of the vertebrae. Using both types of fusion arecontemplated.

In certain embodiments, the disclosure relates to methods of growingbone at a desired area by locally administering a pharmaceuticalformulation containing a compound disclosed herein such as clotrimazole,honokiol, magnolol, tacrolimus, pimecrolimus, sirolimus, everolimus,temsirolimus, spironolactone, fluticasone, fluticasone propionate,fluticasone furoate, linezolid, telmisartan, chlorambucil, retinol,isotretinoin, acitretin, etretinate, retinoic acid (tretinoin),teniposide, mitomycin C, cytarabine, decitabine, vinblastine,vincristine, vindesine, vinorelbine, valrubicin, doxorubicin,daunorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone,plicamycin, pazopanib, topotecan, camptothecin, irinotecan, sunitinib,derivatives, or salts thereof, to the area of desired bone growth withor without the presence of a graft composition or bone growth matrixoptionally in combination with a growth factor such as a bonemorphogenetic protein. In certain embodiments, the disclosurecontemplates administering the pharmaceutical compositions betweenvertebra, e.g., in the area usually occupied by in the intervertebraldisc, to form a spinal fusion.

Cartilage Repair

Cartilage is typically composed of chondroblasts, Type I and Type IIcollagen fibers, elastin fibers, and proteoglycans. Typical locationswithin the human body to find cartilage are the joints between bones,the ear, the nose, the elbow, the knee, the ankle, and theintervertebral discs. Cartilage can become damaged because of trauma ordisease. In some embodiments, the disclosure relates to using compoundsdisclosed herein such as clotrimazole, honokiol, magnolol, tacrolimus,pimecrolimus, sirolimus, everolimus, temsirolimus, spironolactone,fluticasone, fluticasone propionate, fluticasone furoate, linezolid,telmisartan, chlorambucil, retinol, isotretinoin, acitretin, etretinate,retinoic acid (tretinoin), teniposide, mitomycin C, cytarabine,decitabine, vinblastine, vincristine, vindesine, vinorelbine,valrubicin, doxorubicin, daunorubicin, epirubicin, idarubicin,mitoxantrone, pixantrone, plicamycin, pazopanib, topotecan,camptothecin, irinotecan, sunitinib, derivatives, or salts thereof forthe repair or regeneration of cartilage such as articular cartilagerepair or regeneration or intervertebral disc cartilage repair orregeneration.

Articular cartilage repair is typically done to restore the cartilage onthe surface of a bone, i.e., hyaline cartilage. Osteochondrialautografts or allografts may be performed. In certain embodiments, thedisclosure contemplates methods of cartilage repair comprisingtransplanting sections of cartilage and/or bone to a location wherecartilage and/or bone was removed and placing a compound disclosedherein such as herein such as clotrimazole, honokiol, magnolol,tacrolimus, pimecrolimus, sirolimus, everolimus, temsirolimus,spironolactone, fluticasone, fluticasone propionate, fluticasonefuroate, linezolid, telmisartan, chlorambucil, retinol, isotretinoin,acitretin, etretinate, retinoic acid (tretinoin), teniposide, mitomycinC, cytarabine, decitabine, vinblastine, vincristine, vindesine,vinorelbine, valrubicin, doxorubicin, daunorubicin, epirubicin,idarubicin, mitoxantrone, pixantrone, plicamycin, pazopanib, topotecan,camptothecin, irinotecan, sunitinib, derivatives, or salts thereof aboutthe surrounding area, e.g., by injections at the site oftransplantation. Bone with its cartilage covering may be removed fromthe same or a different joint and replanted into the hole left fromremoving degraded bone and cartilage. The transplanted bone andcartilage are typically taken from areas of low stress.

In autologous chondrocyte implantation, cartilage cells are typicallyextracted arthroscopically from normal articular cartilage of thesubject that is located in a nonload-bearing area, e.g., theintercondylar notch or the superior ridge of the femoral condyles, andthe cells are replicated, in vitro, in the presence of growth factors.In certain embodiments, the disclosure relates to replicating cartilagecells comprising mixing hyaline cartilage and a compound disclosedherein such as clotrimazole, honokiol, magnolol, tacrolimus,pimecrolimus, sirolimus, everolimus, temsirolimus, spironolactone,fluticasone, fluticasone propionate, fluticasone furoate, linezolid,telmisartan, chlorambucil, retinol, isotretinoin, acitretin, etretinate,retinoic acid (tretinoin), teniposide, mitomycin C, cytarabine,decitabine, vinblastine, vincristine, vindesine, vinorelbine,valrubicin, doxorubicin, daunorubicin, epirubicin, idarubicin,mitoxantrone, pixantrone, plicamycin, pazopanib, topotecan,camptothecin, irinotecan, sunitinib, derivatives, or salts thereof,under conditions such that the cartilage cells replicate. Typically thisis done by adding other growth factors to the cartilage replicatingmedium, e.g., cartilage-derived morphogenetic proteins and/or BMPproteins. The replicated chondrocytes are implanted to the desired area,e.g., injected about the site of the area for repair optionally incombination with either a membrane or a matrix comprising growth factorssuch as a CDMP, BMP protein or a compound disclosed herein.

Repair of articular cartilage may be performed by marrow stimulatingprocedures sometimes referred to as microfracture surgery. Damagedcartilage is typically ablated by, e.g., drilling or pounding, exposingthe underlying bone sometimes referred to as a microfracture. Thesubchondal bone typically generates a blood clot followed by cartilageregeneration. In some embodiments the disclosure relates to methods ofgenerating cartilage by disrupting bone underlying articular cartilageand placing a compound disclosed herein about the area of disruption,e.g., by injecting compounds disclosed herein such as clotrimazole,honokiol, magnolol, tacrolimus, pimecrolimus, sirolimus, everolimus,temsirolimus, spironolactone, fluticasone, fluticasone propionate,fluticasone furoate, linezolid, telmisartan, chlorambucil, retinol,isotretinoin, acitretin, etretinate, retinoic acid (tretinoin),teniposide, mitomycin C, cytarabine, decitabine, vinblastine,vincristine, vindesine, vinorelbine, valrubicin, doxorubicin,daunorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone,plicamycin, pazopanib, topotecan, camptothecin, irinotecan, sunitinib,derivatives, or salts thereof about the site of disrupted bone for theimproved repair or regeneration of cartilage optionally in combinationwith a growth factor such as a CDMP and/or BMP protein. Alternatively itis contemplated that the compounds are administered to the subject in apharmaceutical composition before, during or after the procedure. Inanother alternative, it is contemplated that a collagen matrix isimplanted at the site of the exposed underlying bone to improvechondrogenic differentiation of mesenchymal stem cells. It is alsocontemplated that the subject may optionally be postoperative injectedwith compounds disclosed herein, hyaluronic acid, and/or mesenchymalstem cells, e.g., obtained from autologous peripheral blood progenitorcells.

Inflammation of the synovial membrane in a joint causes swelling andjoint surface destruction. Removing excess fluid and material by alavage or debridement frequently resolves arthritic knee inflammationand pain. In certain embodiments, the disclosure relates to the use ofcompounds disclosed herein such as clotrimazole, honokiol, magnolol,tacrolimus, pimecrolimus, sirolimus, everolimus, temsirolimus,spironolactone, fluticasone, fluticasone propionate, fluticasonefuroate, linezolid, telmisartan, chlorambucil, retinol, isotretinoin,acitretin, etretinate, retinoic acid (tretinoin), teniposide, mitomycinC, cytarabine, decitabine, vinblastine, vincristine, vindesine,vinorelbine, valrubicin, doxorubicin, daunorubicin, epirubicin,idarubicin, mitoxantrone, pixantrone, plicamycin, pazopanib, topotecan,camptothecin, irinotecan, sunitinib, derivatives, or salts thereofbefore, during, or after a lavage or debridement inside a joint, e.g.,arthroscopic lavage, arthroscopic debridement. In arthroscopicdebridement, joint material or degenerative cartilage it typicallyremoved by injecting a fluid and removing it with a vacuum.

An intervertebral disc (IVD) is found in between two vertebrae. The IVDcontains different tissue types such as the annulus fibrosus (AF), thenucleus pulposus (NP), and end-plates. The AF is made up of mainlycollagen type I. The amount of collagen type I decreases and collagentype II increase gradually nearer the NP which is mostly collagen typeII dispersed within a proteoglycan-rich gelatinous matrix surroundingthe NP.

Porous silk scaffolds may be used for a variety of tissue-engineeringapplications, such as the regeneration of bone and cartilage. Removal ofsericin from silk reduces immunogenic responses. Silk may form a desiredsponge-like structure by freeze-drying a silk solution. Bone marrowmesenchymal stem cells (BMSC) may be incorporated into porous silkscaffolds wrapped around a silicone NP substitute to form an artificialintervertebral disc. In certain embodiments, it is contemplated thatcompounds disclosed herein may be used to generate a matrix of annulusfibrosus by mixing with mesenchymal stem cells and growth factors. Incertain embodiments, the disclosure contemplates implanting a fabricatedintervertebral disc into a subject wherein the disc comprises annulusfibrosus tissue and placing a compound disclosed herein about the siteof the implant location, e.g., by injection, optionally in combinationwith a growth factor such as a cartilage-derived morphogenetic protein(CDMP), e.g., CDMP-1 or CDMP-2, and/or bone morphogenetic proteins,e.g., BMP-7 or BMP-14. The fabricated disc may comprise a NP area with ahydrogel polymer/copolymer matrix or a collagen and/or hyaluronan and/orchondroitin-6-sulfate copolymer. A variety of stem cells, such asmesenchymal stem cells, synovium-derived stem cells (SDSCs), ornotochord cells, may be used for rejuvenation of NP cells.

Therapeutic Applications

In some embodiments, the disclosure relates to pharmaceuticalcompositions comprising compounds disclosed herein for therapeuticapplications. In some embodiments, the disclosure relates to methods oftreating bone degenerative disorders, such as osteoporosis, osteitisdeformans (“Paget's disease of bone”), bone metastasis (with or withouthypercalcaemia), multiple myeloma, primary hyperparathyroidism, orosteogenesis imperfecta. Osteoporosis is a disease of bones that leadsto an increased risk of fracture. In osteoporosis, the bone mineraldensity (BMD) is reduced, bone microarchitecture is disrupted, and theamount and variety of proteins in bone is altered. Osteoporosis is mostcommon in women after menopause, when it is called postmenopausalosteoporosis, but may also develop in men, and may occur in anyone inthe presence of particular hormonal disorders and other chronic diseasesor as a result of medications, specifically glucocorticoids, when thedisease is called steroid- or glucocorticoid-induced osteoporosis (SIOPor GIOP).

Osteoporotic fractures are those that occur in situations where healthypeople would not normally break a bone; they are therefore regarded asfragility fractures. Typical fragility fractures occur in the vertebralcolumn, rib, hip and wrist. The diagnosis of osteoporosis can be madeusing conventional radiography by measuring the bone mineral density(BMD).

In some embodiments, the disclosure relates to treating bonedegenerative disorders by administering pharmaceutical compositiondescribed herein in combination with other agents, such as calciumcarbonate and calcium citrate, vitamin D, cholecalciferol,1,25-dihydroxycholecalciferol, calcitriol, estrogen, testosterone,raloxifene, pamidronate, neridronate, olpadronate, alendronate(Fosamax), ibandronate (Boniva), risedronate (Actonel), zoledronate(Zometa, Aclasta), etidronate (Didronel), clodronate (Bonefos, Loron),or tiludronate (Skelid).

In some embodiments, the disclosure relates to using compounds disclosedherein such as clotrimazole, honokiol, magnolol, tacrolimus,pimecrolimus, sirolimus, everolimus, temsirolimus, spironolactone,fluticasone, fluticasone propionate, fluticasone furoate, linezolid,telmisartan, chlorambucil, retinol, isotretinoin, acitretin, etretinate,retinoic acid (tretinoin), teniposide, mitomycin C, cytarabine,decitabine, vinblastine, vincristine, vindesine, vinorelbine,valrubicin, doxorubicin, daunorubicin, epirubicin, idarubicin,mitoxantrone, pixantrone, plicamycin, pazopanib, topotecan,camptothecin, irinotecan, sunitinib, derivatives, or salts thereof inthe treatment of chondrodystrophies. Typically an effective amount of apharmaceutical composition comprising the compound is administered to asubject diagnosed with, at risk of, or exhibiting symptoms ofosteoarthritis, achondroplasia, costochondrits, relapsingpolychondritis, or articular cartilage damage. The pharmaceuticalcompositions may provide pain relief or slow down the progression ofdamage delaying joint replacement (knee replacement) surgery.

In some embodiments, the disclosure relates to using compounds disclosedherein such as clotrimazole, honokiol, magnolol, tacrolimus,pimecrolimus, sirolimus, everolimus, temsirolimus, spironolactone,fluticasone, fluticasone propionate, fluticasone furoate, linezolid,telmisartan, chlorambucil, retinol, isotretinoin, acitretin, etretinate,retinoic acid (tretinoin), teniposide, mitomycin C, cytarabine,decitabine, vinblastine, vincristine, vindesine, vinorelbine,valrubicin, doxorubicin, daunorubicin, epirubicin, idarubicin,mitoxantrone, pixantrone, plicamycin, pazopanib, topotecan,camptothecin, irinotecan, sunitinib, derivatives, or salts thereof inthe treatment of a degenerative intervertebrial disc. Typically aneffective amount of a pharmaceutical composition comprising the compoundis administered to a subject diagnosed with, at risk of, or exhibitingsymptoms of a degenerative disc. The compositions may be administeredorally or injected directly into an intervertebral disc (IVD), e.g.,into the annulus fibrosus (AF) and/or the nucleus pulposus (NP)optionally in combination with a growth factor such as acartilage-derived morphogenetic protein (CDMP), e.g., CDMP-1 or CDMP-2,or a bone morphogenetic protein, e.g., BMP-7 or BMP-14.

Formulations

Pharmaceutical compositions disclosed herein may be in the form ofpharmaceutically acceptable salts, as generally described below. Somepreferred, but non-limiting examples of suitable pharmaceuticallyacceptable organic and/or inorganic acids are hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, acetic acid and citricacid, as well as other pharmaceutically acceptable acids known per se(for which reference is made to the references referred to below).

When the compounds of the disclosure contain an acidic group as well asa basic group, the compounds of the disclosure may also form internalsalts, and such compounds are within the scope of the disclosure. Whenthe compounds of the disclosure contain a hydrogen-donating heteroatom(e.g. NH), the disclosure also covers salts and/or isomers formed bytransfer of said hydrogen atom to a basic group or atom within themolecule.

Pharmaceutically acceptable salts of the compounds include the acidaddition and base salts thereof. Suitable acid addition salts are formedfrom acids which form non-toxic salts. Examples include the acetate,adipate, aspartate, benzoate, besylate, bicarbonate/carbonate,bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate,esylate, formate, fumarate, gluceptate, gluconate, glucuronate,hexafluorophosphate, hibenzate, hydrochloride/chloride,hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate,maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate,nicotinate, nitrate, orotate, oxalate, palmitate, pamoate,phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate,saccharate, stearate, succinate, tannate, tartrate, tosylate,trifluoroacetate and xinofoate salts. Suitable base salts are formedfrom bases which form non-toxic salts. Examples include the aluminium,arginine, benzathine, calcium, choline, diethylamine, diolamine,glycine, lysine, magnesium, meglumine, olamine, potassium, sodium,tromethamine and zinc salts. Hemisalts of acids and bases may also beformed, for example, hemisulphate and hemicalcium salts. For a review onsuitable salts, see Handbook of Pharmaceutical Salts: Properties,Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002), incorporatedherein by reference.

The compounds described herein may be administered in the form ofprodrugs. A prodrug can include a covalently bonded carrier whichreleases the active parent drug when administered to a mammaliansubject. Prodrugs can be prepared by modifying functional groups presentin the compounds in such a way that the modifications are cleaved,either in routine manipulation or in vivo, to the parent compounds.Prodrugs include, for example, compounds wherein a hydroxy group isbonded to any group that, when administered to a mammalian subject,cleaves to form a free hydroxy group. Examples of prodrugs include, butare not limited to, acetate, formate, and benzoate derivatives ofalcohol functional groups in the compounds. Methods of structuring acompound as prodrugs can be found in the book of Testa and Mayer,Hydrolysis in Drug and Prodrug Metabolism, Wiley (2006). Typicalprodrugs form the active metabolite by transformation of the prodrug byhydrolytic enzymes, the hydrolysis of amide, lactams, peptides,carboxylic acid esters, epoxides or the cleavage of esters of inorganicacids.

Pharmaceutical compositions for use in the present disclosure typicallycomprise an effective amount of a compound and a suitable pharmaceuticalacceptable carrier. The preparations may be prepared in a manner knownper se, which usually involves mixing the at least one compoundaccording to the disclosure with the one or more pharmaceuticallyacceptable carriers, and, if desired, in combination with otherpharmaceutical active compounds, when necessary under asepticconditions. Reference is made to U.S. Pat. Nos. 6,372,778, 6,369,086,6,369,087 and 6,372,733 and the further references mentioned above, aswell as to the standard handbooks, such as the latest edition ofRemington's Pharmaceutical Sciences.

Generally, for pharmaceutical use, the compounds may be formulated as apharmaceutical preparation comprising at least one compound and at leastone pharmaceutically acceptable carrier, diluent or excipient and/oradjuvant, and optionally one or more further pharmaceutically activecompounds.

The pharmaceutical preparations of the disclosure are preferably in aunit dosage form, and may be suitably packaged, for example in a box,blister, vial, bottle, sachet, ampoule or in any other suitablesingle-dose or multi-dose holder or container (which may be properlylabeled); optionally with one or more leaflets containing productinformation and/or instructions for use. Generally, such unit dosageswill contain between 1 and 1000 mg, and usually between 5 and 500 mg, ofthe at least one compound of the disclosure, e.g. about 10, 25, 50, 100,200, 300 or 400 mg per unit dosage.

The compounds can be administered by a variety of routes including theoral, ocular, rectal, transdermal, subcutaneous, intravenous,intramuscular or intranasal routes, depending mainly on the specificpreparation used. The compound will generally be administered in an“effective amount”, by which is meant any amount of a compound that,upon suitable administration, is sufficient to achieve the desiredtherapeutic or prophylactic effect in the subject to which it isadministered. Usually, depending on the condition to be prevented ortreated and the route of administration, such an effective amount willusually be between 0.01 to 1000 mg per kilogram body weight of thepatient per day, more often between 0.1 and 500 mg, such as between 1and 250 mg, for example about 5, 10, 20, 50, 100, 150, 200 or 250 mg,per kilogram body weight of the patient per day, which may beadministered as a single daily dose, divided over one or more dailydoses. The amount(s) to be administered, the route of administration andthe further treatment regimen may be determined by the treatingclinician, depending on factors such as the age, gender and generalcondition of the patient and the nature and severity of thedisease/symptoms to be treated. Reference is again made to U.S. Pat.Nos. 6,372,778, 6,369,086, 6,369,087, and 6,372,733 and the furtherreferences mentioned above, as well as to the standard handbooks, suchas the latest edition of Remington's Pharmaceutical Sciences.

For an oral administration form, the compound can be mixed with suitableadditives, such as excipients, stabilizers or inert diluents, andbrought by means of the customary methods into the suitableadministration forms, such as tablets, coated tablets, hard capsules,aqueous, alcoholic, or oily solutions. Examples of suitable inertcarriers are gum arabic, magnesia, magnesium carbonate, potassiumphosphate, lactose, glucose, or starch, in particular, corn starch. Inthis case, the preparation can be carried out both as dry and as moistgranules. Suitable oily excipients or solvents are vegetable or animaloils, such as sunflower oil or cod liver oil. Suitable solvents foraqueous or alcoholic solutions are water, ethanol, sugar solutions, ormixtures thereof. Polyethylene glycols and polypropylene glycols arealso useful as further auxiliaries for other administration forms. Asimmediate release tablets, these compositions may containmicrocrystalline cellulose, dicalcium phosphate, starch, magnesiumstearate and lactose and/or other excipients, binders, extenders,disintegrants, diluents and lubricants known in the art.

When administered by nasal aerosol or inhalation, the compositions maybe prepared according to techniques well-known in the art ofpharmaceutical formulation and may be prepared as solutions in saline,employing benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons, and/or othersolubilizing or dispersing agents known in the art. Suitablepharmaceutical formulations for administration in the form of aerosolsor sprays are, for example, solutions, suspensions or emulsions of thecompounds of the disclosure or their physiologically tolerable salts ina pharmaceutically acceptable solvent, such as ethanol or water, or amixture of such solvents. If required, the formulation can alsoadditionally contain other pharmaceutical auxiliaries such assurfactants, emulsifiers and stabilizers as well as a propellant.

For subcutaneous or intravenous administration, the compounds, ifdesired with the substances customary therefore such as solubilizers,emulsifiers or further auxiliaries are brought into solution,suspension, or emulsion. The compounds of formula I can also belyophilized and the lyophilizates obtained used, for example, for theproduction of injection or infusion preparations. Suitable solvents are,for example, water, physiological saline solution or alcohols, e.g.ethanol, propanol, glycerol, sugar solutions such as glucose or mannitolsolutions, or mixtures of the various solvents mentioned. The injectablesolutions or suspensions may be formulated according to known art, usingsuitable non-toxic, parenterally-acceptable diluents or solvents, suchas mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodiumchloride solution, or suitable dispersing or wetting and suspendingagents, such as sterile, bland, fixed oils, including synthetic mono- ordiglycerides, and fatty acids, including oleic acid.

When rectally administered in the form of suppositories, theformulations may be prepared by mixing the compounds of formula I with asuitable non-irritating excipient, such as cocoa butter, syntheticglyceride esters or polyethylene glycols, which are solid at ordinarytemperatures, but liquefy and/or dissolve in the rectal cavity torelease the drug.

In certain embodiments, it is contemplated that these compositions canbe extended release formulations. Typical extended release formationsutilize an enteric coating. Typically, a barrier is applied to oralmedication that controls the location in the digestive system where itis absorbed. Enteric coatings prevent release of medication before itreaches the small intestine. Enteric coatings may contain polymers ofpolysaccharides, such as maltodextrin, xanthan, scleroglucan dextran,starch, alginates, pullulan, hyaloronic acid, chitin, chitosan and thelike; other natural polymers, such as proteins (albumin, gelatin etc.),poly-L-lysine; sodium poly(acrylic acid);poly(hydroxyalkylmethacrylates) (for example poly(hydroxyethylmethacrylate)); carboxypolymethylene (for example Carbopol™); carbomer;polyvinyl pyrrolidone; gums, such as guar gum, gum arabic, gum karaya,gum ghatti, locust bean gum, tamarind gum, gellan gum, gum tragacanth,agar, pectin, gluten and the like; poly(vinyl alcohol); ethylene vinylalcohol; polyethylene glycol (PEG); and cellulose ethers, such ashydroxymethylcellulose (HMC), hydroxyethylcellulose (HEC),hydroxypropylcellulose (HPC), methylcellulose (MC), ethylcellulose (EC),carboxyethylcellulose (CEC), ethylhydroxyethyl cellulose (EHEC),carboxymethylhydroxyethylcellulose (CMHEC),hydroxypropylmethyl-cellulose (HPMC), hydroxypropylethylcellulose (HPEC)and sodium carboxymethylcellulose (Na CMC); as well as copolymers and/or(simple) mixtures of any of the above polymers. Certain of theabove-mentioned polymers may further be crosslinked by way of standardtechniques.

The choice of polymer will be determined by the nature of the activeingredient/drug that is employed in the composition of the disclosure aswell as the desired rate of release. In particular, it will beappreciated by the skilled person, for example in the case of HPMC, thata higher molecular weight will, in general, provide a slower rate ofrelease of drug from the composition. Furthermore, in the case of HPMC,different degrees of substitution of methoxyl groups and hydroxypropoxylgroups will give rise to changes in the rate of release of drug from thecomposition. In this respect, and as stated above, it may be desirableto provide compositions of the disclosure in the form of coatings inwhich the polymer carrier is provided by way of a blend of two or morepolymers of, for example, different molecular weights in order toproduce a particular required or desired release profile.

Microspheres of polylactide, polyglycolide, and their copolymerspoly(lactide-co-glycolide) may be used to form sustained-release proteinor compound delivery systems. Proteins and/or compounds can be entrappedin the poly(lactide-co-glycolide) microsphere depot by a number ofmethods, including formation of a water-in-oil emulsion with water-borneprotein and organic solvent-borne polymer (emulsion method), formationof a solid-in-oil suspension with solid protein dispersed in asolvent-based polymer solution (suspension method), or by dissolving theprotein in a solvent-based polymer solution (dissolution method). Onecan attach poly(ethylene glycol) to proteins (PEGylation) to increasethe in vivo half-life of circulating therapeutic proteins and decreasethe chance of an immune response.

EXPERIMENTAL Compound Screenings

Compounds were selected by virtual screening for development based oncomputational modeling, docking, and in silico screening. The structuraldata revealed that the surfaces of BMPs have binding epitopes forBMPR-IA interaction involving the main chain amide groups of amino acidsL51 and D53 with minor contribution from the hydrophobic interactions.Two hydrogen bonds are formed between L51 (main chain amide andcarbonyl) of BMP-2 and Q86 of BMPR-IA. The core structure of BMPR-IIshares the same pattern of disulfide connectivity as ActR-II, withdisulfide bonds. In an earlier analysis a hypothetical complex betweenBMPR-II and BMP2, created by superimposing BMPR-II in the position ofActR-II in the ternary complex between ActR-II, BMP2, and BMPR-IA,suggested that BMPR-II makes similar overall ligand binding contacts toBMP2 as does ActR-II. From Residues Tyr67, Trp85, and Phel15 of BMPRIIare in the main hydrophobic patch and Lys81, Ser86, Glu93, and Tyr113are also important binding determinants. In addition, His87 and Tyr40confer specificity in BMPR-II ligand binding. Mutational analysis hasidentified A34, H39, S88, L90, and L100 residues as binding determinantsof BMP-2 for BMPR-II and ActR-II. See Sebald et al., Biol Chem, 2004,385(8):697-710.

Crystal structure of BMPR-II was superimposed on to the ActR-IIB ofternary complex consisting of BMP-2 dimer/BMPR-IAEC/ActR-IIB using a webbased tool Superpose. Using these superposed structures a newhypothetical ternary complex consisting of BMP-2/BMPR-IAEC/BMPR-II wasmodelled. The surface area (A2) for each residue was calculated and thepercent solvent accessible contact area (% SA) of the model ternarycomplex of BMP-2/BMPR-IAEC/BMPR-II and individual monomer structures,and the position (Pos) and name of the amino acid residues in thebinding interface of both the receptors, BMPR-IA and BMPR-II that bindto BMP-2 were identified.

LUDI de novo design method of Accelrys Discovery Studios 3.0 and alsoNCC library of compounds, which contains Phase I, II, and III drugs,were used against the receptor regions that bind to BMP. The BMP bindingregion of receptors is divided into ten sub regions (with someoverlapping residues) for LUDI runs. The binding site defined as spheresof radius 10 angstrom covering the amino acids in the regions. The LUDIde novo design method against these ten regions derived 300 novel smallmolecules that were chemically synthesized for cell-based screeningassays. LUDI against the BMPR-IA and BMPR-II regions that bind to BMP-2were also used to obtain molecules from NCC library that are drugmolecules from Phase I, II or III.

The BMP-potentiating activities of compounds may be evaluated bymonitoring several markers of the osteoblastic phenotype correspondingto various time points during phenotype differentiation of C2C12 cellstowards terminally differentiated osteoblasts.

Cell Culture

Mouse C2C12 cells and Dulbecco's modified Eagle's medium (DMEM) werepurchased from ATCC (Manassas, Va.). The non-heat inactivated fetalbovine serum (FBS) was purchased from HyClone Laboratories, Inc. (Logan,Utah). The C2C12 cells at passages 5 to 10 were subcultured in T-75 cm²flasks in DMEM supplemented with 10% FBS at 37° C. in 5% CO₂ withhumidification. When the flasks reached 80% confluence, the cells weretrypsinized and seeded in triplicate.

Alkaline Phosphatase (ALP) Assay

The C2C12 cells were plated at 200,000 cells/well in 6-well plates andgrown overnight in DMEM containing 10% FBS. On day 2, the culture mediumwas replaced with DMEM containing 2% FBS and the cells were treated withvarious concentrations of compound for 24 hours. On day 3, the culturemedium was replaced with DMEM containing 2% FBS and the cells weretreated with 50 ng/ml of BMP-2 and various concentrations of compoundfor 72 hours. The cells were washed with phosphate-buffered saline (PBS)and lysed by addition of lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM MgCl₂and 1.0% Triton X-100). The cell lysates were centrifuged for 5 minutesat 13,000×g. The supernatant was removed and the aliquots were assayedfor ALP activity and protein amount. The ALP activity was measured intriplicate using an ALP assay kit (Sigma-Aldrich, St. Louis, Mo.) inmicrotiter plates. The protein amount was determined with Bio-Radprotein assay reagent (Bio-Rad, Hercules, Calif.) using bovine serumalbumin (BSA) as a standard. The ALP activity (nmoles of p-nitrophenolper ml) was normalized to the protein amount (nmoles of p-nitrophenolper μg).

BMP-Noggin Competitive Alkaline Phosphatase (ALP) Assay

The C2C12 cells were plated at 200,000 cells/well in 6-well plates andgrown overnight in DMEM containing 10% FBS. On day 2, the culture mediumwas replaced with DMEM containing 2% FBS and the cells were treated with90-100 ng/ml of BMP-2 (as noted in each experiment), variousconcentrations of compound, and 100-120 ng/ml of noggin (as noted ineach experiment) for 72 hours. The cells were washed withphosphate-buffered saline (PBS) and lysed by addition of lysis buffer(10 mM Tris-HCl pH 8.0, 1 mM MgCl₂ and 1.0% Triton X-100). The celllysates were centrifuged for 5 minutes at 13,000×g. The supernatant wasremoved and the aliquots were assayed for ALP activity and proteinamount. The ALP activity was measured in triplicate using an ALP assaykit (Sigma-Aldrich, St. Louis, Mo.) in microtiter plates. The proteinamount was determined with Bio-Rad protein assay reagent (Bio-Rad,Hercules, Calif.) using bovine serum albumin (BSA) as a standard. TheALP activity (nmoles of p-nitrophenol per ml) was normalized to theprotein amount (nmoles of p-nitrophenol per μg).

Collagen Disc Implantation with Macrolides in Rat Ectopic Model

Sprague Dawley rats about 5-6 weeks of age were chest implanted with acollagen disc (diameter of 1.0 cm and height of 2.0 mm; total volume of150 cubic millimeters) and doses of macrolides in the absence of BMP-2.After 4 weeks the rats were sacrificed and evaluated for bone growth.See table below. De novo bone formation is demonstrated locally withsirolimus, everolimus, and tacrolimus. In certain embodiments, thedisclosure contemplates local bone formation for fracture repair,segmental bone defects, spine fusion, bone grafting, and regional boneenhancement for osteopenic bones before they fracture (e.g. hip,vertebral body, etc) by deliver locally to induce local bone formation.

Animal # with Dose Xray: Drug (mM) Volume Carrier 1 . . . 2 . . . 3 . .. 4 Results Ave. Sirolimus 0 100 ul collagen 0, 0, 0, 0 No bone (0 of 4)0 10 100 ul collagen 0, (1), 0, 0 Bone (1 of 4) 0.25 15 100 ul collagen0, 0, 0, (2) Bone (1 of 4) 0.5 20 100 ul collagen 0, 0, (3), 0 Bone (1of 4) 0.75 0 100 ul collagen 0, (1), 0, 0 Bone (1 of 4) 0.25 25 100 ulcollagen (1), 5, 0, 5 Bone (3 of 4) 2.75 30 100 ul collagen x, 4, 5, 5Bone (3 of 4) 3.5 35 100 ul collagen 3, 5, 4, 5 Bone (4 of 4) 4.25Everolimus 0 100 ul collagen 0, 0, 0, 0 No bone (0 of 4) 0 10 100 ulcollagen 0, 0, 0, 0 No bone (0 of 4) 0 15 100 ul collagen 5, 0, (2), 3Bone (3 of 4) 2.5 20 100 ul collagen 4, 3, 4, 4 Bone (4 of 4) 3.75 0 100ul collagen 0, 0, 0, 0 No bone (0 of 4) 0 25 100 ul collagen 5, 5, 5,4Bone (4 of 4) 4.75 30 100 ul collagen 5, 5, x, 5 Bone (3 of 4) 3.75 35100 ul collagen 4, 5, 5, 5 Bone (4 of 4) 4.75 Tacrolimus 0 100 ulcollagen 0, 0, 0, 0 No bone (0 of 4) 0 10 100 ul collagen 0, 0, 0, 0 Nobone (0 of 4) 0 25 100 ul collagen 4, 5, 4, 4 Bone (4 of 4) 4.25 35 100ul collagen 2, 5, 4, 5 Bone (4 of 4) 4 0 100 ul collagen 0, 0, 0, 0 Nobone (0 of 4) 0 15 100 ul collagen 0, 2, 0, 0 Bone (1 of 4) 0.5 20 100ul collagen 0, 5, 6, 2 Bone (3 of 4) 3.25 30 100 ul collagen 0, 3, 5, 2Bone (3 of 4) 2.5

The invention claimed is:
 1. A method of forming bone in vivo comprisingimplanting a graft composition comprising calcium phosphates andtacrolimus in a subject such that bone forms in the graft, wherein thegraft composition does not contain a growth factor.
 2. The method ofclaim 1, wherein more than 0.7 mg of tacrolimus is present per 150 mm³of bone graft volume.
 3. The method of claim 1, wherein said calciumphosphates are hydroxyapatite and tricalcium phosphate.
 4. A method ofperforming spinal fusion comprising implanting a bone graft compositioncomprising collagen or hydrogel matrix and tacrolimus in a subject suchthat bone forms in the graft between two vertebrae of a subject, whereinthe graft composition does not contain a growth factor.
 5. The method ofclaim 4, wherein the subject has symptoms of back pain.
 6. The method ofclaim 4, wherein the subject is diagnosed with a degenerative disc. 7.The method of claim 4, wherein more than 0.7 mg of tacrolimus is presentper 150 mm³ of bone graft volume.
 8. The method of claim 4, wherein thebone graft composition comprises a further comprises calcium phosphates.9. The method of claim 8, wherein said calcium phosphates arehydroxyapatite and tricalcium phosphate.
 10. A method of performingspinal fusion comprising implanting a bone graft composition comprisingcalcium phosphates and tacrolimus in a subject such that bone forms inthe graft between two vertebrae of a subject, wherein the graftcomposition does not contain a growth factor.
 11. The method of claim10, wherein the subject has symptoms of back pain.
 12. The method ofclaim 10, wherein the subject is diagnosed with a degenerative disc. 13.The method of claim 10, wherein more than 0.7 mg of tacrolimus ispresent per 150 mm³ of bone graft volume.
 14. The method of claim 10,wherein said calcium phosphates are hydroxyapatite and tricalciumphosphate.